Target molecule attachment to surfaces

ABSTRACT

Method and reagent composition for covalent attachment of target molecules, such as nucleic acids, onto the surface of a substrate. The reagent composition includes groups capable of covalently binding to the target molecule. Optionally, the composition can contain photoreactive groups for use in attaching the reagent composition to the surface. The reagent composition can be used to provide activated slides for use in preparing microarrays of nucleic acids.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 08/940,213, for “Reagent and Method for AttachingTarget Molecules to a Surface”, filed Sep. 30, 1997, now U.S. Pat. No.5,858,653, the entire disclosure of which is incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to methods for attaching target moleculessuch as oligonucleotides (oligos) to a surface, and to compositions foruse in such methods. In another aspect, the invention relates to theresultant coated surfaces themselves. In yet another aspect, theinvention relates to the use of photochemical and thermochemical meansto attach molecules to a surface.

BACKGROUND OF THE INVENTION

The immobilization of deoxyribonucleic acid (DNA) onto support surfaceshas become an important aspect in the development of DNA-based assaysystems as well as for other purposes, including the development ofmicrofabricated arrays for DNA analysis. See, for instance, “MicrochipArrays Put DNA on the Spot”, R. Service, Science 282(5388):396-399, 16October 1998; and “Fomenting a Revolution, in Miniature”, I. Amato,Science 282(5388): 402-405, Oct. 16, 1998.

See also, “The Development of Microfabricated Arrays of DNA Sequencingand Analysis”, O'Donnell-Maloney et al., TIBTECH 14:401-407 (1996).Generally, such procedures are carried out on the surface of microwellplates, tubes, beads, microscope slides, silicon wafers or membranes.Certain approaches, in particular, have been developed to enable orimprove the likelihood of end-point attachment of a syntheticoligonucleotide to a surface. End-point attachment (i.e., with thenucleic acid sequence attached through one or the other terminalnucleotide) is desirable because the entire length of the sequence willbe available for hybridization to another nucleic acid sequence. This isparticularly advantageous for the detection of single base pair changesunder stringent hybridization conditions.

Hybridization is the method used most routinely to measure nucleic acidsby base pairing to probes immobilized on a solid support. When combinedwith amplification techniques such as the polymerase chain reaction(PCR) or ligase chain reaction (LCR), hybridization assays are apowerful tool for diagnosis and research. Microwell plates, inparticular, are convenient and useful for assaying relatively largenumbers of samples. Several methods have been used for immobilization ofnucleic acid probes onto microwell plates. Some of these involveadsorption of unmodified or modified oligonucleotides onto polystyreneplates. Others involve covalent immobilization. Various methods havealso been used to increase the sensitivity of hybridization assays.Polymeric capture probes (also known as target molecules) and detectionprobes have been synthesized and used to obtain sensitivities down to10⁷ DNA molecules/ml. Another method used branched oligonucleotides toincrease the sensitivity of hybridization assays. Yet another methodused a multi-step antibody-enhanced method. Other types of nucleic acidprobes such as ribonucleic acid (RNA), complementary DNA (cDNA) andpeptide nucleic acids (PNA's) have also been immobilized onto microwellplates for hybridization of PCR products in diagnostic applications.Furthermore, PCR primers have been immobilized onto microwell plates forsolid phase PCR.

Only a relative few approaches to immobilizing DNA, to date, have foundtheir way into commercial products. One such product is known as“NucleoLink™”, and is available from Nalge Nunc International (see,e.g., Nunc Tech Note Vol. 3, No. 17). In this product, the DNA isreacted with a carbodiimide to activate 5′-phosphate groups which thenreact with functional groups on the surface. Disadvantages of thisapproach are that it requires the extra step of adding the carbodiimidereagent as well as a five hour reaction time for immobilization of DNA,and it is limited to a single type of substrate material.

As another example, Pierce has recently introduced a proprietary DNAimmobilization product known as “Reacti-Bind™ DNA Coating Solutions”(see “Instructions—Reacti-Bind™ DNA Coating Solution” 1/1997). Thisproduct is a solution that is mixed with DNA and applied to surfacessuch as polystyrene or polypropylene. After overnight incubation, thesolution is removed, the surface washed with buffer and dried, afterwhich it is ready for hybridization. Although the product literaturedescribes it as being useful for all common plastic surfaces used in thelaboratory, it does have some limitations. For example, Applicants werenot able to demonstrate useful immobilization of DNA onto polypropyleneusing the manufacturer's instructions. Furthermore, this productrequires large amounts of DNA. The instructions indicate that the DNAshould be used at a concentration between 0.5 and 5 μg/ml.

Similarly, Costar sells a product called “DNA-BINDTM” for use inattaching DNA to the surface of a well in a microwell plate (see, e.g.,the DNA-BINDTM “Application Guide”). The surface of the DNA-BIND™ plateis coated with an uncharged, nonpolymeric heterobifunctional reagentcontaining an N-oxysuccinimide (NOS) reactive group. This group reactswith nucleophiles such as primary amines. The heterobifunctional coatingreagent also contains a photochemical group and spacer arm whichcovalently links the reactive group to the surface of the polystyreneplate. Thereafter, amine-modified DNA can be covalently coupled to theNOS surface. The DNA is modified by adding a primary amine either duringthe synthesis process to the nascent oligomer or enzymatically to thepreformed sequence. Since the DNA-BIND™ product is polystyrene based, itis of limited use for those applications that require elevatedtemperatures such as thermal cycling.

These various products may be useful for some purposes, or under certaincircumstances, but all tend to suffer from one or more drawbacks andconstraints. In particular, they either tend to require large amounts ofoligonucleotide, render background noise levels that are unsuitably highand/or lack versatility.

International Patent Application No. PCT/US98/20140, assigned to theassignee of the present application, describes and claims, inter alia, areagent composition for attaching a target molecule to the surface of asubstrate, the reagent composition comprising one or more groups forattracting the target molecule to the reagent, and one or morethermochemically reactive groups for forming covalent bonds withcorresponding functional groups on the attracted target molecule.Optionally, the composition further provides photogroups for use inattaching the composition to a surface. In one embodiment, for instance,a plurality of photogroups and a plurality of cationic groups (in theform of quaternary ammonium groups) are attached to a hydrophilicpolymer backbone. This polymer can then be coimmobilized with a secondpolymer backbone that provides the above-described thermochemicallyreactive groups (e.g., N-oxysuccinimide (“NOS”) groups) forimmobilization of target molecules.

While reagent compositions having both attracting groups andthermochemically reactive groups, as described in the above-captionedPCT application, remain useful and preferred for many applications,Applicants also find that the attracting groups may not be requiredunder all circumstances. For instance, one suitable process forpreparing activated slides for microarrays includes the steps of coatingthe slides with a reagent composition of a type described in the PCTapplication (and particularly, one having both attracting groups as wellas photoreactive and thermochemically reactive groups). The polymers areattached to the slide by activation of the photoreactive groups,following by the application of small volumes (e.g., several nanolitersor less) of target molecules (e.g., oligonucleotides) using precisionprinting techniques.

Once applied, the solvent used to deliver the oligonucleotide is dried(as the oligonucleotides are attracted to the bound polymer), and theslide incubated under conditions suitable to permit the thermochemicalcoupling of the oligonucleotide to the bound polymer. Thereafter,however, any unbound oligonucleotide is typically washed off of theslide. Applicants have found, however, that there occasionally remains adetectable trail of unbound oligonucleotide, referred to as a “cometeffect”, leading away from the spot. This trail is presumably due to theattractive forces within the bound polymer present on the slide surfacethat surrounds the spot, serving to tie up the generally negativelycharged oligonucleotide as it is washed from the spot. This trail, inturn, can provide undesirable and unduly high levels of backgroundnoise.

Applicants have found that under such circumstances (e.g., theapplication of small volumes directly to a generally flat surface)polymeric reagents are preferably provided without the presence of suchattracting groups (though with the thermochemically reactive groups andoptional photogroups). Suitable reagents of this type are disclosed inthe above-captioned co-pending PCT application. Such reagents, in turn,can be used to coat oligonucleotides in a manner that provides animproved combination of such properties as reduced background, smallspot size (e.g., increased contact angle), as compared to polymericreagents having charged attracting groups.

SUMMARY OF THE INVENTION

The present invention provides a method and reagent composition forcovalent attachment of target molecules onto the surface of a substrate,such as microwell plates, tubes, beads, microscope slides, siliconwafers or membranes. In one embodiment, the method and composition areused to immobilize nucleic acid probes onto plastic materials such asmicrowell plates, e.g., for use in hybridization assays. In a preferredembodiment the method and composition are adapted for use withsubstantially flat surfaces, such as those provided by microscope slidesand other plastic, silicon hydride, or organosilane-pretreated glass orsilicone slide support surfaces. The reagent composition can then beused to covalently attach a target molecule such as a biomolecule (e.g.,a nucleic acid) which in turn can be used for specific binding reactions(e.g., to hybridize a nucleic acid to its complementary strand).

Support surfaces can be prepared from a variety of materials, includingbut not limited to plastic materials selected from the group consistingof crystalline thermoplastics (e.g., high and low density polyethylenes,polypropylenes, acetal resins, nylons and thermoplastic polyesters) andamorphous thermoplastics (e.g., polycarbonates and poly(methylmethacrylates). Suitable plastic or glass materials provide a desiredcombination of such properties as rigidity, toughness, resistance tolong term deformation, recovery from deformation on release of stress,and resistance to thermal degradation.

A reagent composition of the invention contains one or morethermochemically reactive groups (i.e., groups having a reaction ratedependent on temperature). Suitable groups are selected from the groupconsisting of activated esters (e.g., NOS), epoxide, azlactone,activated hydroxyl and maleimide groups. Optionally, and preferably, thecomposition can also contain one or more photoreactive groups.Additionally, the reagent may comprise one or more hydrophilic polymers,to which the thermochemically reactive and/or photoreactive groups canbe pendent. The photoreactive groups (alternatively referred to hereinas “photogroups”) can be used, for instance, to attach reagent moleculesto the surface of the support upon the application of a suitable energysource such as light. The thermochemically reactive groups, in turn, canbe used to form covalent bonds with appropriate and complementaryfunctional groups on the target molecule.

Generally, the reagent molecules will first be attached to the surfaceby activation of the photogroups, thereafter the target molecule, (e.g.,an oligonucleotide) is contacted with the bound reagent under conditionssuitable to permit it to come into binding proximity with the boundpolymer. The target molecule is thermochemically coupled to the boundreagent by reaction between the reactive groups of the bound reagent andappropriate functional groups on the target molecule. Thethermochemically reactive groups and the ionic groups can either be onthe same polymer or, for instance, on different polymers that arecoimmobilized onto the surface. Optionally, and preferably, the targetmolecule can be prepared or provided with functional groups tailored togroups of the reagent molecule. During their synthesis, for instance,the oligonucleotides can be prepared with functional groups such asamines or sulfhydryl groups.

The invention further provides a method of attaching a target molecule,such as an oligo, to a surface, by employing a reagent as describedherein. In turn, the invention provides a surface having nucleic acidsattached thereto by means of such a reagent, as well as a material(e.g., microwell plate) that provides such a surface. In yet anotheraspect, the invention provides a composition comprising a reagent(s) ofthis invention in combination with a target molecule that contains oneor more functional groups reactive with the thermochemically reactivegroup(s) of the reagent.

Using such reagents, applicants have found that capture probes can becovalently immobilized to a variety of surfaces, including surfaces thatwould not otherwise adsorb the probes (such as polypropylene andpolyvinylchloride). The resulting surfaces provide signals comparable toor better than those obtained with modified oligonucleotides adsorbedonto polystyrene or polycarbonate.

The present immobilization reagent and method can be used inamplification methods in a manner that is simpler than those previouslyreported, and can also provide improved surfaces for the covalentimmobilization of nucleophile-derivatized nucleic acids. In addition toimmobilized probes for amplification methods and hybridization assays,the reagents of this invention may provide improved nucleic acidimmobilization for solid phase sequencing and for immobilizing primersfor PCR and other amplification techniques.

DETAILED DESCRIPTION

A preferred reagent molecule of the present invention comprises ahydrophilic backbone bearing one or more thermochemically reactivegroups useful for forming a covalent bond with the correspondingfunctional group of the target molecule, together with one or morephotoreactive groups useful for attaching the reagent to a surface.

In another embodiment of the invention, it is possible to immobilizenucleic acid sequences without the use of the photoreactive group. Forinstance, the surface of the material to be coated can be provided withthermochemically reactive groups, which can be used to immobilizehydrophilic polymers having thermochemically reactive groups asdescribed above. For example, a surface may be treated with an ammoniaplasma to introduce a limited number of reactive amines on the surfaceof the material. If this surface is then treated with a hydrophilicpolymer having thermochemically reactive groups (e.g., NOS groups), thenthe polymer can be immobilized through reaction of the NOS groups withcorresponding amine groups on the surface. Preferably, the reactivegroups on the polymer are in excess relative to the correspondingreactive groups on the surface to insure that a sufficient number ofthese thermochemically reactive groups remain following theimmobilization to allow coupling with the nucleic acid sequence.

While not intending to be bound by theory, it appears that by virtue ofthe small spot size, as well as the kinetics and fluid dynamicsencountered in the use of reduced spot sizes, the oligonucleotide isable to come into binding proximity with the bound reagent without theneed for the attracting groups described above. When used for preparingmicroarrays, e.g., to attach capture molecules (e.g., oligonucleotidesor cDNA) to the microarray surface, such capture molecules are generallydelivered to the surface in a volume of less than about 1 nanoliter perspot, using printing pins adapted to form the spots into arrays havingcenter to center spacing of about 200 μm to about 500 μm.

Given their small volumes, the printed target arrays tend to dryquickly, thus further affecting the coupling kinetics and efficiency.Unlike the coupling of DNA from solution and onto the surface of coatedmicroplate wells, oligonucleotides printed in arrays of extremely smallspot sizes tend to dry quickly, thereby altering the parametersaffecting the manner in which the oligonucleotides contact and couplewith the support. In addition to the design and handling of the printingpins, other factors can also affect the spot size, and in turn, theultimate hybridization signals, including: salt concentrations, type ofsalts and wetting agents in the printing buffer; hydrophobic/hydrophilicproperties of the surfaces; the size and/or concentration of theoligonucleotide; and the drying environments.

As described herein (e.g., in Examples 25, 26 and 28), coatings ofreagents having both photogroups and thermochemically reactive groups(“Photo-PA-PolyNOS”), as well as reagents having those groups togetherwith attracting groups (a mixture of“Photo-PA-PolyNOS/Photo-PA-PolyQuat”) both provided useful and specificimmobilization of amine-modified DNA, with the choice between the twoapproaches being largely dependent on the choice of substrate (e.g.,flat slide as opposed to microwell).

In a preferred embodiment, the reagent composition can be used toprepare activated slides having the reagent composition photochemicallyimmobilized thereon. The slides can be stably stored and used at a laterdate to prepare microarrays by immobilizing amine-modified DNA. Thecoupling of the capture DNA to the surface takes place at pH 8-9 in ahumid environment following printing the DNA solution in the form ofsmall spots.

Activated slides of the present invention are particularly well suitedto replace conventional (e.g., silylated) glass slides in thepreparation of microarrays using manufacturing and processing protocols,reagents and equipment such as micro-spotting robots (e.g., as availablefrom Cartesian), and a chipmaker micro-spotting device (e.g., asavailable from TeleChem International). Suitable spotting equipment andprotocols are commercially available, such as the “Arraylt”™ ChipMaker 3spotting device. This product is said to represent an advanced versionof earlier micro-spotting technology, employing 48 printing pins todeliver as many as 62,000 samples per solid substrate.

The use of such an instrument, in combination with conventional (e.g.,poly-1-lysine coated) slides, is well known in the art. See, forinstance, U.S. Pat. No. 5,087,522 (Brown et al.) “Methods forFabricating Microarrays of Biological Samples”, and the references citedtherein, the disclosures of each of which are incorporated herein byreference.

For instance, the method and system of the present invention can be usedto provide a substrate, such as a glass slide, with a surface having oneor more microarrays. Each microarray preferably provides at least about100/cm² (and preferably at least about 1 000/cm²) distinct targetmolecules (e.g., polynucleotide or polypeptide biopolymers) in a surfacearea of less than about 1 cm². Each distinct target molecule (1) isdisposed at a separate, defined position in the array, (2) has a lengthof at least 10 subunits, (3) is present in a defined amount betweenabout 0.1 femtomoles and about 10 nanomoles, and (4) is deposited inselected volume in the volume range of about 0.01 nanoliters to about100 nanoliters. These regions (e.g., discrete spots) within the arraycan be generally circular in shape, with a typical diameter of betweenabout 10 microns and about 500 microns (and preferably between about 20and about 200 microns). The regions are also preferably separated fromother regions in the array by about the same distance (e.g., center tocenter spacing of about 20 microns to about 1000 microns) . A pluralityof analyte-specific regions can be provided, such that each regionincludes a single, and preferably different, analyte specific reagent(“target molecule”).

Those skilled in the art, given the present description, will be able toidentify and select suitable reagents depending on the type of targetmolecule of interest. Target molecules include, but are not limited to,plasmid DNA, cosmid DNA, bacteriophage DNA, genomic DNA (includes, butnot limited to yeast, viral, bacterial, mammalian, insect), RNA, cDNA,PNA, and oligonucleotides.

A polymeric backbone can be either synthetic or naturally occurring, andis preferably a synthetic polymer selected from the group consisting ofoligomers, homopolymers, and copolymers resulting from addition orcondensation polymerization. Naturally occurring polymers, such aspolysaccharides, polypeptides can be used as well. Preferred backbonesare biologically inert, in that they do not provide a biologicalfunction that is inconsistent with, or detrimental to, their use in themanner described.

Such polymer backbones can include acrylics such as those polymerizedfrom hydroxyethyl acrylate, hydroxyethyl methacrylate, glycerylacrylate, glyceryl methacrylate, acrylamide and methacrylamide, vinylssuch as polyvinyl pyrrolidone and polyvinyl alcohol, nylons such aspolycaprolactam, polylauryl lactam, polyhexamethylene adipamide andpolyhexamethylene dodecanediamide, polyurethanes and polyethers (e.g.,polyethylene oxides).

The polymeric backbones of the invention are chosen to providehydrophilic backbones capable of bearing the desired number and type ofthermochemically reactive groups, and optionally photogroups, thecombination dependent upon the reagent selected. The polymeric backboneis also selected to provide a spacer between the surface and thethermochemically reactive groups. In this manner, the reagent can bebonded to a surface or to an adjacent reagent molecule, to provide theother groups with sufficient freedom of movement to demonstrate optimalactivity. The polymer backbones are preferably hydrophilic (e.g., watersoluble), with polyacrylamide and polyvinylpyrrolidone beingparticularly preferred polymers.

Reagents of the invention carry one or more pendent latent reactive(preferably photoreactive) groups covalently bound (directly orindirectly) to the polymer backbone. Photoreactive groups are definedherein, and preferred groups are sufficiently stable to be stored underconditions in which they retain such properties. See, e.g., U.S. Pat.No. 5,002,582, the disclosure of which is incorporated herein byreference. Latent reactive groups can be chosen that are responsive tovarious portions of the electromagnetic spectrum, with those responsiveto ultraviolet and visible portions of the spectrum (referred to hereinas “photoreactive”) being particularly preferred.

Photoreactive groups respond to specific applied external stimuli toundergo active specie generation with resultant covalent bonding to anadjacent chemical structure, e.g., as provided by the same or adifferent molecule. Photoreactive groups are those groups of atoms in amolecule that retain their covalent bonds unchanged under conditions ofstorage but that, upon activation by an external energy source, formcovalent bonds with other molecules.

The photoreactive groups generate active species such as free radicalsand particularly nitrenes, carbenes, and excited states of ketones uponabsorption of electromagnetic energy. Photoreactive groups may be chosento be responsive to various portions of the electromagnetic spectrum,and photoreactive groups that are responsive to e.g., ultraviolet andvisible portions of the spectrum are preferred and may be referred toherein occasionally as “photochemical group” or “photogroup”.

Photoreactive aryl ketones are preferred, such as acetophenone,benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles(i.e., heterocyclic analogs of anthrone such as those having N, O, or Sin the 10-position), or their substituted (e.g., ring substituted)derivatives. The functional groups of such ketones are preferred sincethey are readily capable of undergoing theactivation/inactivation/reactivation cycle described herein.Benzophenone is a particularly preferred photoreactive moiety, since itis capable of photochemical excitation with the initial formation of anexcited singlet state that undergoes intersystem crossing to the tripletstate. The excited triplet state can insert into carbon-hydrogen bondsby abstraction of a hydrogen atom (from a support surface, for example),thus creating a radical pair. Subsequent collapse of the radical pairleads to formation of a new carbon-carbon bond. If a reactive bond(e.g., carbon-hydrogen) is not available for bonding, the ultravioletlight-induced excitation of the benzophenone group is reversible and themolecule returns to ground state energy level upon removal of the energysource. Photoactivatible aryl ketones such as benzophenone andacetophenone are of particular importance inasmuch as these groups aresubject to multiple reactivation in water and hence provide increasedcoating efficiency. Hence, photoreactive aryl ketones are particularlypreferred.

The azides constitute a preferred class of photoreactive groups andinclude arylazides (C₆R₅N₃) such as phenyl azide and particularly4-fluoro-3-nitrophenyl azide, acyl azides (—CO—N₃) such as benzoyl azideand p-methylbenzoyl azide, azido formates (—O—CO—N₃) such as ethylazidoformate, phenyl azidoformate, sulfonyl azides (—SO₂—N₃) such asbenzenesulfonyl azide, and phosphoryl azides (RO)₂PON₃ such as diphenylphosphoryl azide and diethyl phosphoryl azide. Diazo compoundsconstitute another class of photoreactive groups and includediazoalkanes (—CHN₂) such as diazomethane and diphenyldiazomethane,diazoketones (—CO—CHN₂) such as diazoacetophenone and1-trifluoromethyl-1-diazo-2-pentanone, diazoacetates (—O—CO—CHN₂) suchas t-butyl diazoacetate and phenyl diazoacetate, andbeta-keto-alpha-diazoacetates (—CO—CN₂—CO—O—) such as t-butyl alphadiazoacetoacetate. Other photoreactive groups include the diazirines(—CHN₂) such as 3-trifluoromethyl-3-phenyldiazirine, and ketenes(—CH=C=O) such as ketene and diphenylketene. Photoactivatible arylketones such as benzophenone and acetophenone are of particularimportance inasmuch as these groups are subject to multiple reactivationin water and hence provide increased coating efficiency.

Upon activation of the photoreactive groups, the reagent molecules arecovalently bound to each other and/or to the material surface bycovalent bonds through residues of the photoreactive groups. Exemplaryphotoreactive groups, and their residues upon activation, are shown asfollows.

Residue Photoreactive Group Functionality aryl axides amine R—NH—R″ acylazides amide R—CO—NH—R″ azidiformates carbamate R—O—CO—NH—R″ sulfonylazides sulfonamide R—SO₂—NH—R″ phosphoryl azides phosphoramide(RO)₂PO—NH—R″ diazoalkanes new C—C bond diazoketones new C—C bond andketone diazoacetates new C—C bond and ester beta-keto-alpha- new C—Cbond and diazoacetates beta-ketoester aliphatic azo new C—C bonddiazirines new C—C bond ketenes new C—C bond photoactivated ketones newC—C bond and alcohol

Those skilled in the art, given the present description, will be able toidentify and select suitable thermochemically reactive groups to providefor covalent immobilization of appropriately derivatized nucleic acidsequences. For example, an amino derivatized nucleic acid sequence willundergo a covalent coupling reaction with an activated ester such as aNOS ester to provide an amide linking group. Similar activated esterssuch p-nitrophenyl and pentafluorophenyl esters would also provide amidelinks when reacted with amine groups. Those skilled in the art wouldalso recognize numerous other amine-reactive functional groups such asisocyanates, thioisocyanates, carboxylic acid chlorides, epoxides,aldehydes, alkyl halides and sulfonate esters, such as mesylate,tosylate and tresylate, each of which could serve as thethermochemically reactive group.

In another example, the nucleic acid sequence can be derivatized with asulfhydryl group using techniques well known in the art. Thecorresponding thermochemically reactive group would be, for example, amaleimide ring structure or an a-iodoacetamide. Either of thesestructures would react readily to provide a covalent linkage with thesulfhydryl derivatized nucleic acid sequence.

The functionalized polymers of this invention can be prepared byappropriate derivatization of a preformed polymer or, more preferably,by polymerization of a set of comonomers to give the desiredsubstitution pattern. The latter approach is preferred because of theease of changing the ratio of the various comonomers and by the abilityto control the level of incorporation into the polymer. A combination ofthese two approaches can also be used to provide optimal structures.

In a preferred embodiment, for instance, monomers are prepared having apolymerizable group at one end of the molecule, separated by a spacergroup from a photoreactive or thermochemically reactive group at theother end. For example, polymerizable vinyl groups such as acrylamides,acrylates, or maleimides can be coupled through a short hydrocarbonspacer to an activated ester such as a NOS ester or to a photoreactivegroup such as a substituted benzophenone. These compounds can beprepared and purified using organic synthesis techniques well known tothose skilled in the art. Some of desired monomers are commerciallyavailable, such as MAPTAC, N-[3-(dimethylamino)propyl]methacrylamide(DMAPMA), and N-(3-aminopropyl)methacrylamide hydrochloride (APMA),these compounds providing quaternary ammonium salts, tertiary amines,and primary amines respectively along the backbone of the polymer.

Polymers and copolymers can be prepared from the above monomers as well,using techniques known to those skilled in the art. Preferably, thesemonomers and copolymers undergo free radical polymerization of vinylgroups using azo initiators such as 2,2′-azobisisobutyronitrile (AIBN)or peroxides such as benzoyl peroxide. The monomers selected for thepolymerization are chosen based on the nature of the final polymerproduct. For example, a photoreactive polymer containing a NOS group isprepared from a monomer containing the photoreactive group and a secondmonomer containing the activated NOS ester.

The composition of the final polymer can be controlled by mole ratio ofthe monomers charged to the polymerization reaction. Typically thesefunctionalized monomers are used at relatively low mole percentages ofthe total monomer content of the polymerization reaction with theremainder of the composition consisting of a monomer which is neitherphotoreactive nor thermochemically reactive toward the nucleic acidsequence. Examples of such monomers include, but are not limited to,acrylamide and N-vinylpyrrolidone. Based on the relative reactivities ofthe monomers used, the distribution of the monomers along the backboneis largely random.

In some cases, the thermochemically reactive group on the backbone ofthe polymer can itself act as a polymerizable monomer, if present duringpolymerization, thus requiring the introduction of that group in asecond step following the initial formation of the polymer. For example,the preparation of a photoreactive polymer having maleimide along thebackbone can be accomplished by an initial preparation of a polymercontaining both photoreactive groups and amine groups using thetechniques described above, followed by reaction of the amine groupswith a heterobifunctional molecule containing a maleimide group and anisocyanate connected by a short hydrocarbon spacer. A wide variety ofsuch polymer modification techniques are available using typical organicreactions known to those skilled in the art.

The invention will be further described with reference to the followingnon-limiting Examples. It will be apparent to those skilled in the artthat many changes can be made in the embodiments described withoutdeparting from the scope of the present invention. Thus the scope of thepresent invention should not be limited to the embodiments described inthis application, but only by embodiments described by the language ofthe claims and the equivalents of those embodiments. Unless otherwiseindicated, all percentages are by weight. Structures of the various“Compounds” identified throughout these Examples can be found in Table13 included below. NMR analyses were run on a 80 Mhz spectrometer unlessotherwise stated.

EXAMPLES Example 1 Preparation of 4-Benzoylbenzoyl Chloride (BBA-C1)(Compound I)

4-Benzoylbenzoic acid (BBA), 1.0 kg (4.42 moles), was added to a dry 5liter Morton flask equipped with reflux condenser and overhead stirrer,followed by the addition of 645 ml (8.84 moles) of thionyl chloride and725 ml of toluene. Dimethylformamide, 3.5 ml, was then added and themixture was heated at reflux for 4 hours. After cooling, the solventswere removed under reduced pressure and the residual thionyl chloridewas removed by three evaporations using 3×500 ml of toluene. The productwas recrystallized from 1:4 toluene:hexane to give 988 g (91 % yield)after drying in a vacuum oven. Product melting point was 92-94° C.Nuclear magnetic resonance (NMR) analysis at 80 MHz (¹H NMR (CDCl₃)) wasconsistent with the desired product: aromatic protons 7.20-8.25 (m, 9H).All chemical shift values are in ppm downfield from a tetramethylsilaneinternal standard. The final compound was stored for use in thepreparation of a monomer used in the synthesis of photoactivatablepolymers as described, for instance, in Example 3.

Example 2 Preparation of N-(3-Aminopropyl)methacrylamide Hydrochloride(APMA) (Compound II)

A solution of 1,3-diaminopropane, 1910 g (25.77 moles), in 1000 ml ofCH₂Cl₂ was added to a 12 liter Morton flask and cooled on an ice bath. Asolution of t-butyl phenyl carbonate, 1000 g (5.15 moles), in 250 ml ofCH₂Cl₂ was then added dropwise at a rate which kept the reactiontemperature below 15° C. Following the addition, the mixture was warmedto room temperature and stirred 2 hours. The reaction mixture wasdiluted with 900 ml of CH₂Cl₂ and 500 g of ice, followed by the slowaddition of 2500 ml of 2.2 N NaOH. After testing to insure the solutionwas basic, the product was transferred to a separatory funnel and theorganic layer was removed and set aside as extract #1. The aqueous wasthen extracted with 3×1250 ml of CH₂Cl₂, keeping each extraction as aseparate fraction. The four organic extracts were then washedsuccessively with a single 1250 ml portion of 0.6 N NaOH beginning withfraction #1 and proceeding through fraction #4. This wash procedure wasrepeated a second time with a fresh 1250 ml portion of 0.6 N NaOH. Theorganic extracts were then combined and dried over Na₂SO₄. Filtrationand evaporation of solvent to a constant weight gave 825 g ofN-mono-t-BOC-1,3-diaminopropane which was used without furtherpurification.

A solution of methacrylic anhydride, 806 g (5.23 moles), in 1020 ml ofCHCl₃ was placed in a 12 liter Morton flask equipped with overheadstirrer and cooled on an ice bath. Phenothiazine, 60 mg, was added as aninhibitor, followed by the dropwise addition ofN-mono-t-BOC-1,3-diaminopropane, 825 g (4.73 moles), in 825 ml of CHCl₃.The rate of addition was controlled to keep the reaction temperaturebelow 10° C. at all times. After the addition was complete, the ice bathwas removed and the mixture was left to stir overnight. The product wasdiluted with 2400 ml of water and transferred to a separatory finnel.After thorough mixing, the aqueous layer was removed and the organiclayer was washed with 2400 ml of 2 N NaOH, insuring that the aqueouslayer was basic. The organic layer was then dried over Na₂SO₄ andfiltered to remove drying agent. A portion of the CHCl₃ solvent wasremoved under reduced pressure until the combined weight of the productand solvent was approximately 3000 g. The desired product was thenprecipitated by slow addition of 11.0 liters of hexane to the stirredCHCl₃ solution, followed by overnight storage at 4° C. The product wasisolated by filtration and the solid was rinsed twice with a solventcombination of 900 ml of hexane and 150 ml of CHCl₃. Thorough drying ofthe solid gave 900 g ofN-[N′-(t-butyloxycarbonyl)-3-aminopropyl]-methacrylamide, m.p. 85.8° C.by DSC. Analysis on an NMR spectrometer was consistent with the desiredproduct: ¹H NMR (CDCl₃) amide NH's 6.30-6.80, 4.55-5.10 (m, 2H), vinylprotons 5.65, 5.20 (m, 2H), methylenes adjacent to N 2.90-3.45 (m, 4H),methyl 1.95 (m, 3H), remaining methylene 1.50-1.90 (m, 2H), and t-butyl1.40 (s, 9H).

A 3-neck, 2 liter round bottom flask was equipped with an overheadstirrer and gas sparge tube. Methanol, 700 ml, was added to the flaskand cooled on an ice bath. While stirring, HCl gas was bubbled into thesolvent at a rate of approximately 5 liters/minute for a total of 40minutes. The molarity of the final HCl/MeOH solution was determined tobe 8.5 M by titration with 1 N NaOH using phenolphthalein as anindicator. The N-[N′-(t-butyloxycarbonyl)-3-aminopropyl]methacrylamide,900 g (3.71 moles), was added to a 5 liter Morton flask equipped with anoverhead stirrer and gas outlet adapter, followed by the addition of1150 ml of methanol solvent. Some solids remained in the flask with thissolvent volume. Phenothiazine, 30 mg, was added as an inhibitor,followed by the addition of 655 ml (5.57 moles) of the 8.5 M HCl/MeOHsolution. The solids slowly dissolved with the evolution of gas but thereaction was not exothermic. The mixture was stirred overnight at roomtemperature to insure complete reaction. Any solids were then removed byfiltration and an additional 30 mg of phenothiazine were added. Thesolvent was then stripped under reduced pressure and the resulting solidresidue was azeotroped with 3×1000 ml of isopropanol with evaporationunder reduced pressure. Finally, the product was dissolved in 2000 ml ofrefluxing isopropanol and 4000 ml of ethyl acetate were added slowlywith stirring. The mixture was allowed to cool slowly and was stored at4° C. overnight. Compound II was isolated by filtration and was dried toconstant weight, giving a yield of 630 g with a melting point of 124.7°C. by DSC. Analysis on an NMR spectrometer was consistent with thedesired product: ¹H NMR (D₂O) vinyl protons 5.60, 5.30 (m, 2H),methylene adjacent to amide N 3.30 (t, 2H), methylene adjacent to amineN 2.95 (t, 2H), methyl 1.90 (m, 3H), and remaining methylene 1.65-2.10(m, 2H). The final compound was stored for use in the preparation of amonomer used in the synthesis of photoactivatable polymers as described,for instance, in Example 3.

Example 3 Preparation of N-[3-(4-Benzoylbenzamido)propyl]methacrylamide(BBA-APMA) (Compound III)

Compound II 120 g (0.672 moles), prepared according to the generalmethod described in Example 2, was added to a dry 2 liter, three-neckround bottom flask equipped with an overhead stirrer. Phenothiazine,23-25 mg, was added as an inhibitor, followed by 800 ml of chloroform.The suspension was cooled below 10° C. on an ice bath and 172.5 g (0.705moles) of Compound I, prepared according to the general method describedin Example 1, were added as a solid. Triethylamine, 207 ml (1.485moles), in 50 ml of chloroform was then added dropwise over a 1-1.5 hourtime period. The ice bath was removed and stirring at ambienttemperature was continued for 2.5 hours. The product was then washedwith 600 ml of 0.3 N HCl and 2×300 ml of 0.07 N HCl. After drying oversodium sulfate, the chloroform was removed under reduced pressure andthe product was recrystallized twice from 4:1 toluene: chloroform using23-25 mg of phenothiazine in each recrystallization to preventpolymerization. Typical yields of Compound III were 90% with a meltingpoint of 147-151° C. Analysis on an NMR spectrometer was consistent withthe desired product: ¹H NMR (CDCl₃) aromatic protons 7.20-7.95 (m, 9H),amide NH 6.55 (broad t, 1H), vinyl protons 5.65, 5.25 (m, 2H), methyleneadjacent to amide N's 3.20-3.60 (m, 4H), methyl 1.95 (s, 3H), andremaining methylene 1.50-2.00 (m, 2H). The final compound was stored foruse in the synthesis of photoactivatable polymers as described, forinstance, in Examples 9-11.

Example 4 Preparation of N-Succinimidyl 6-Maleimidohexanoate(MAL-EAC-NOS) (Compound IV)

A functionalized monomer was prepared in the following manner, and wasused as described in Examples 9 and 12 to introduce activated estergroups on the backbone of a polymer. 6-Aminohexanoic acid, 100 g (0.762moles), was dissolved in 300 ml of acetic acid in a three-neck, 3 literflask equipped with an overhead stirrer and drying tube. Maleicanhydride, 78.5 g (0.801 moles), was dissolved in 200 ml of acetic acidand added to the 6-aminohexanoic acid solution. The mixture was stirredone hour while heating on a boiling water bath, resulting in theformation of a white solid. After cooling overnight at room temperature,the solid was collected by filtration and rinsed with 2×50 ml of hexane.After drying, the typical yield of the (Z)-4-oxo-5-aza-2-undecendioicacid was 158-165 g (90-95%) with a melting point of 160-165° C. Analysison an NMR spectrometer was consistent with the desired product: ¹H NMR(DMSO-d₆) amide proton 8.65-9.05 (m, 1H), vinyl protons 6.10, 6.30 (d,2H), methylene adjacent to nitrogen 2.85-3.25 (m, 2H), methyleneadjacent to carbonyl 2.15 (t, 2H), and remaining methylenes 1.00-1.75(m, 6H).

(Z)-4-Oxo-5-aza-2-undecendioic acid, 150.0 g (0.654 moles), aceticanhydride, 68 ml (73.5 g, 0.721 moles), and phenothiazine, 500 mg, wereadded to a 2 liter three-neck round bottom flask equipped with anoverhead stirrer. Triethylamine, 91 ml (0.653 moles), and 600 ml of THFwere added and the mixture was heated to reflux while stirring. After atotal of 4 hours of reflux, the dark mixture was cooled to <60° C. andpoured into a solution of 250 ml of 12 N HCl in 3 liters of water. Themixture was stirred 3 hours at room temperature and then was filteredthrough a filtration pad (Celite 545, J. T. Baker, Jackson, Tenn.) toremove solids. The filtrate was extracted with 4×500 ml of chloroformand the combined extracts were dried over sodium sulfate. After adding15 mg of phenothiazine to prevent polymerization, the solvent wasremoved under reduced pressure. The 6-maleimidohexanoic acid wasrecrystallized from 2:1 hexane:chloroform to give typical yields of76-83 g (55-60%) with a melting point of 81-85° C. Analysis on a NMRspectrometer was consistent with the desired product: ¹H NMR (CDCl₃)maleimide protons 6.55 (s, 2H), methylene adjacent to nitrogen 3.40 (t,2H), methylene adjacent to carbonyl 2.30 (t, 2H), and remainingmethylenes 1.05-1.85 (m, 6H).

The 6-maleimidohexanoic acid, 20.0 g (94.7 mmol), was dissolved in 100ml of chloroform under an argon atmosphere, followed by the addition of41 ml (0.47 mol) of oxalyl chloride. After stirring for 2 hours at roomtemperature, the solvent was removed under reduced pressure with 4×25 mlof additional chloroform used to remove the last of the excess oxalylchloride. The acid chloride was dissolved in 100 ml of chloroform,followed by the addition of 12 g (0.104 mol) of N-hydroxysuccinimide and16 ml (0.114 mol) of triethylamine. After stirring overnight at roomtemperature, the product was washed with 4×100 ml of water and driedover sodium sulfate. Removal of solvent gave 24 g of product (82%) whichwas used without further purification. Analysis on an NMR spectrometerwas consistent with the desired product: ¹H NMR (CDCl₃) maleimideprotons 6.60 (s, 2H), methylene adjacent to nitrogen 3.45 (t, 2H),succinimidyl protons 2.80 (s, 4H), methylene adjacent to carbonyl 2.55(t, 2H), and remaining methylenes 1.15-2.00 (m, 6H). The final compoundwas stored for use in the synthesis of photoactivatable polymers asdescribed, for instance, in Examples 9 and 12.

Example 5 Preparation of N-Succinimidyl 6-Methacrylamidohexanoate(MA-EAC-NOS) (Compound V)

A functionalized monomer was prepared in the following manner, and wasused as described in Example 11 to introduce activated ester groups onthe backbone of a polymer. 6-Aminocaproic acid, 4.00 g (30.5 mmol), wasplaced in a dry round bottom flask equipped with a drying tube.Methacrylic anhydride, 5.16 g ( 33.5 mmol), was then added and themixture was stirred at room temperature for four hours. The resultingthick oil was triturated three times with hexane and the remaining oilwas dissolved in chloroform, followed by drying over sodium sulfate.After filtration and evaporation, a portion of the product was purifiedby silica gel flash chromatography using a 10% methanol in chloroformsolvent system. The appropriate fractions were combined, 1 mg ofphenothiazine was added, and the solvent was removed under reducedpressure. Analysis on an NMR spectrometer was consistent with thedesired product: ¹H NMR (CDCl₃) carboxylic acid proton 7.80-8.20 (b,1H), amide proton 5.80-6.25 (b, 1H), vinyl protons 5.20 and 5.50 (m,2H), methylene adjacent to nitrogen 3.00-3.45 (m, 2H), methyleneadjacent to carbonyl 2.30 (t, 2H), methyl group 1.95 (m, 3H), andremaining methylenes 1.10-1.90 (m, 6H).

6-Methacrylamidohexanoic acid, 3.03 g (15.2 mmol), was dissolved in 30ml of dry chloroform, followed by the addition of 1.92 g (16.7 mmol) ofN-hydroxysuccinimide and 6.26 g (30.4 mmol) of1,3-dicyclohexylcarbodiimide. The reaction was stirred under a dryatmosphere overnight at room temperature. The solid was then removed byfiltration and a portion was purified by silica gel flashchromatography. Non-polar impurities were removed using a chloroformsolvent, followed by elution of the desired product using a 10%tetrahydrofuran in chloroform solvent. The appropriate fractions werepooled, 0.2 mg of phenothiazine were added, and the solvent wasevaporated under reduced pressure. This product, containing smallamounts of 1,3-dicyclohexylurea as an impurity, was used without furtherpurification. Analysis on an NMR spectrometer was consistent with thedesired product: ¹H NMR (CDCl₃) amide proton 5.60-6.10 (b, 1H), vinylprotons 5.20 and 5.50 (m, 2H), methylene adjacent to nitrogen 3.05-3.40(m, 2H), succinimidyl protons 2.80 (s, 4H), methylene adjacent tocarbonyl 2.55 (t, 2H), methyl 1.90 (m, 3H), and remaining methylenes1.10-1.90 (m, 6H). The final compound was stored for use in thesynthesis of photoactivatable polymers as described, for instance, inExample 11.

Example 6 Preparation of 4-Bromomethylbenzophenone (BMBP)(Compound VI)

4-Methylbenzophenone, 750 g (3.82 moles), was added to a 5 liter Mortonflask equipped with an overhead stirrer and dissolved in 2850 ml ofbenzene. The solution was then heated to reflux, followed by thedropwise addition of 610 g (3.82 moles) of bromine in 330 ml of benzene.The addition rate was approximately 1.5 ml/min and the flask wasilluminated with a 90 watt (90 joule/sec) halogen spotlight to initiatethe reaction . A timer was used with the lamp to provide a 10% dutycycle (on 5 seconds, off 40 seconds), followed in one hour by a 20% dutycycle (on 10 seconds, off 40 seconds). At the end of the addition, theproduct was analyzed by gas chromatography and was found to contain 71%of the desired Compound VI, 8% of the dibromo product, and 20% unreacted4-methylbenzophenone. After cooling, the reaction mixture was washedwith 10 g of sodium bisulfite in 100 ml of water, followed by washingwith 3×200 ml of water. The product was dried over sodium sulfate andrecrystallized twice from 1:3 toluene:hexane. After drying under vacuum,635 g of Compound VI were isolated, providing a yield of 60% and havinga melting point of 112-114° C. Analysis on an NMR spectrometer wasconsistent with the desired product: ¹H NMR (CDCl₃) aromatic protons7.20-7.80 (m, 9H) and benzylic protons 4.48 (s, 2H). The final compoundwas stored for use in the preparation of a photoactivatable chaintransfer agent as described in Example 7.

Example 7 Preparation ofN-(2-Mercaptoethyl)-3.5-bis(4-benzoylbenzyloxy)benzamide (Compound VII)

3,5-Dihydroxybenzoic acid, 46.2 g (0.30 mol), was weighed into a 250 mlflask equipped with a Soxhlet extractor and condenser. Methanol, 48.6ml, and concentrated sulfuric acid, 0.8 ml, were added to the flask and48 g of 3A molecular sieves were placed in the Soxhlet extractor. Theextractor was filled with methanol and the mixture was heated at refluxovernight. Gas chromatographic analysis of the resulting product showeda 98% conversion to the desired methyl ester. The solvent was removedunder reduced pressure to give approximately 59 g of crude product. Theproduct was used in the following step without further purification. Asmall sample was previously purified for NMR analysis, resulting in aspectrum consistent with the desired product: ¹H NMR (DMSO-d₆) aromaticprotons 6.75 (d, 2H) and 6.38 (t, 1H), and methyl ester 3.75 (s, 3H).

The entire methyl ester product from above was placed in a 2 liter flaskwith an overhead stirrer and condenser, followed by the addition of173.25 g (0.63 mol) of Compound VI, prepared according to the generalmethod described in Example 6, 207 g (1.50 mol) of potassium carbonate,and 1200 ml of acetone. The resulting mixture was then refluxedovernight to give complete reaction as indicated by thin layerchromatography (TLC). The solids were removed by filtration and theacetone was evaporated under reduced pressure to give 49 g of crudeproduct. The solids were diluted with 1 liter of water and extractedwith 3×1 liter of chloroform. The extracts were combined with theacetone soluble fraction and dried over sodium sulfate, yielding 177 gof crude product. The product was recrystallized from acetonitrile togive 150.2 g of a white solid, a 90% yield for the first two steps.Melting point of the product was 131.5° C. (DSC) and analysis on an NMRspectrometer was consistent with the desired product: 1H NMR (CDCl₃)aromatic protons 7.25-7.80 (m, 18H), 7.15 (d, 2H), and 6.70 (t, 1H),benzylic protons 5.05 (s, 4H), and methyl ester 3.85 (s, 3H).

The methyl 3,5-bis(4-benzoylbenzyloxy)benzoate, 60.05 g (0.108 mol), wasplaced in a 2 liter flask, followed by the addition of 120 ml of water,480 ml of methanol, and 6.48 g (0.162 mol) of sodium hydroxide. Themixture was heated at reflux for three hours to complete hydrolysis ofthe ester. After cooling, the methanol was removed under reducedpressure and the sodium salt of the acid was dissolved in 2400 ml ofwarm water. The acid was precipitated using concentrated hydrochloricacid, filtered, washed with water, and dried in a vacuum oven to give58.2 g of a white solid (99% yield). Melting point on the product was188.3° C. (DSC) and analysis on an NMR spectrometer was consistent withthe desired product: 1H NMR (DMSO-d6) aromatic protons 7.30-7.80 (m,18H), 7.15 (d, 2H), and 6.90 (t, 1H), and benzylic protons 5.22 (s, 4H).

The 3,5-bis(4-benzoylbenzyloxy)benzoic acid, 20.0 g (36.86 mmol), wasadded to a 250 ml flask, followed by 36 ml of toluene, 5.4 ml (74.0mmol) of thionyl chloride, and 28 μl of N,N-dimethylformamide. Themixture was refluxed for four hours to form the acid chloride. Aftercooling, the solvent and excess thionyl chloride were removed underreduced pressure. Residual thionyl chloride was removed by fouradditional evaporations using 20 ml of chloroform each. The crudematerial was recrystallized from toluene to give 18.45 g of product, an89% yield. Melting point on the product was 126.9° C. (DSC) and analysison an NMR spectrometer was consistent with the desired product: ¹H NMR(CDC1₃) aromatic protons 7.30-7.80 (m, 18H), 7.25 (d, 2H), and 6.85 (t,1H), and benzylic protons 5.10 (s, 4H).

The 2-aminoethanethiol hydrochloride, 4.19 g (36.7 mmol), was added to a250 ml flask equipped with an overhead stirrer, followed by 15 ml ofchloroform and 10.64 ml (76.5 mmol) of triethylamine. After cooling theamine solution on an ice bath, a solution of3,5-bis(4-benzoylbenzyloxy)benzoyl chloride, 18.4 g (32.8 mmol), in 50ml of chloroform was added dropwise over a 50 minute period. Cooling onice was continued 30 minutes, followed by warming to room temperaturefor two hours. The product was diluted with 150 ml of chloroform andwashed with 5×250 ml of 0.1 N hydrochloric acid. The product was driedover sodium sulfate and recrystallized twice from 15:1 toluene: hexaneto give 13.3 g of product, a 67% yield. Melting point on the product was115.9° C. (DSC) and analysis on an NMR spectrometer was consistent withthe desired product.: 1H NMR (DMSO-d₆) aromatic protons 7.20-7.80 (m,18H), 6.98 (d, 2H), and 6.65 (t, 1H), amide NH 6.55 (broad t, 1H),benzylic protons 5.10 (s, 4H), methylene adjacent to amide N 3.52 (q,2H), methylene adjacent to SH 2.10 (q, 2H), and SH 1.38 (t, 1H). Thefinal compound was stored for use as a chain transfer agent in thesynthesis of photoactivatable polymers as described, for instance, inExample 12.

Example 8 Preparation of N-Succinimidyl 11-(4-Benzoylbenzamido)undecanoate (BBA-AUD-NOS) (Compound VIII)

Compound I (50 g, 0.204 mol), prepared according to the general methoddescribed in Example 1, was dissolved in 2500 ml of chloroform, followedby the addition of a solution of 43.1 g (0.214 mol) of11-aminoundecanoic acid and 60.0 g (1.5 mol) of sodium hydroxide in 1500ml of water. The mixture was stirred vigorously for one hour in a 5liter Morton flask to insure thorough mixing of the two layers. Themixture was acidified with 250 ml of concentrated hydrochloric acid andstirred an additional 30 minutes. The organic layer was separated andthe aqueous was extracted with 3×500 ml of chloroform. The combinedorganic extracts were dried over sodium sulfate, filtered, andevaporated to give a solid. The product was recrystallized from tolueneto give 68.37 g (82%) of 11-(4-benzoylbenzamido)undecanoic acid with amelting point of 107-109° C. Analysis on an NMR spectrometer wasconsistent with the desired product: ¹H NMR (CDCI₃) aromatic protons7.20-7.80 (m, 9H), amide NH 6.30 (broad t, 1H), methylene adjacent toamide N 3.35 (m, 2H), methylene adjacent to carbonyl 2.25 (t, 2H), andremaining methylenes 1.00-1.80 (m, 16H).

The 11-(4-benzoylbenzamido)undecanoic acid, 60.0 g (0.146 mol), wasdissolved with warming in 1200 ml of anhydrous 1,4-dioxane in anoven-dried 2000 ml flask. After cooling to room temperature, 17.7 g (0.154 mol) of N-hydroxysuccinimide and 33.2 g (0.161 mol) of1,3-dicyclohexylcarbodiimide were added to the solution and the mixturewas stirred overnight under a dry atmosphere. The solids were thenremoved by filtration, rinsing the filter cake with 1,4-dioxane. Thesolvent was then removed under vacuum and the product was recrystallizedtwice from ethanol. After thorough drying in a vacuum oven, 53.89 g (73% yield) of a white solid were obtained with a melting point of 97-99°C. Analysis on an NMR spectrometer was consistent with the desiredproduct: ¹H NMR (CDCl₃) aromatic protons 7.20-7.80 (m, 9H), amide NH6.25 (broad t, 1H), methylene adjacent to amide N 3.35 (m, 2H),methylenes on succinimidyl ring 2.75 (s, 4H), methylene adjacent tocarbonyl 2.55 (t, 2H), and remaining methylenes 1.00-1.90 (m, 16H).

Example 9 Preparation of Copolymer of Acrylamide. BBA-APMA. andMAL-EAC-NOS (Random Photo PA-PolyNOS) (Compounds IX, A-D)

A photoactivatable copolymer of the present invention was prepared inthe following manner. Acrylamide, 4.298 g (60.5 mmol), was dissolved in57.8 ml of tetrahydrofuran (THF), followed by 0.219 g ( 0.63 mmol) ofCompound III, prepared according to the general method described inExample 3, 0.483 g (1.57 mmol) of Compound IV, prepared according to thegeneral method described in Example 4, 0.058 ml (0.39 mmol) ofN,N,N′,N′-tetramethylethylenediamine (TEMED), and 0.154 g (0.94 mmol) of2,2′-azobisisobutyronitrile (AIBN). The solution was deoxygenated with ahelium sparge for 3 minutes, followed by an argon sparge for anadditional 3 minutes. The sealed vessel was then heated overnight at 60°C. to complete the polymerization. The solid product was isolated byfiltration and the filter cake was rinsed thoroughly with THF and CHCl₃.The product was dried in a vacuum oven at 30° C. to give 5.34 g of awhite solid. NMR analysis (DMSO-d₆) confirmed the presence of the NOSgroup at 2.75 ppm and the photogroup load was determined to be 0.118mmol BBA/g of polymer. The MAL-EAC-NOS composed 2.5 mole % of thepolymerizable monomers in this reaction to give Compound IX-A.

The above procedure was used to prepare a polymer having 5 mole %Compound IV. Acrylamide, 3.849 g (54.1 mmol), was dissolved in 52.9 mlof THF, followed by 0.213 g ( 0.61 mmol) of Compound VI, preparedaccording to the general method described in Example 3, 0.938 g (3.04mmol) of Compound IV, prepared according to the general method describedin Example 4, 0.053 ml (0.35 mmol) of TEMED and 0.142 g (0.86 mmol) ofAIBN. The resulting solid, Compound IX-B, when isolated as describedabove, gave 4.935 g of product with a photogroup load of 0.101 mmolBBA/g of polymer.

The above procedure was used to prepare a polymer having 10 mole %Compound IV. Acrylamide, 3.241 g (45.6 mmol), was dissolved in 46.4 mlof THF, followed by 0.179 g ( 0.51 mmol) of Compound III, preparedaccording to the general method described in Example 3, 1.579 g (5.12mmol) of Compound IV, prepared according to the general method describedin Example 4, 0.047 ml (0.31 mmol) of TEMED and 0.126 g (0.77 mmol) ofAIBN. The resulting solid, Compound IX-C, when isolated as describedabove, gave 4.758 g of product with a photogroup load of 0.098 mmolBBA/g of polymer.

A procedure similar to the above procedure was used to prepare a polymerhaving 2.5 mole % Compound IV and 2 mole % Compound III. Acrylamide,16.43 g (231.5 mmol); Compound III, prepared according to the generalmethod described in Example 3, 1.70 g (4.85 immol); Compound IV,prepared according to the general method described in Example 4, 1.87 g(6.06 mmol); and THF (222 ml) were stirred in a round bottom flask withan argon sparge at room temperature for 15 minutes. TEMED, 0.24 ml (2.14mmol), and AIBN, 0.58 g (3.51 mmol), were added to the reaction. Thereaction was then refluxed for 4 hours under an atmosphere of argon. Theresulting solid, Compound IX-D, when isolated as described above, gave19.4 g of product with a photogroup load of 0.23 mmol BBA/g of polymer.

Example 10 Preparation of Copolymer of Acrylamide, BBA-APMA, and[3-(Methacryloylamino)propyl]trimethylammonium Chloride (Random PhotoPA-PolyQuat) (Compounds X, A-B)

A photoactivatable copolymer of the present invention was prepared inthe following manner. Acrylamide, 10.681 g (0.150 mol), was dissolved in150 ml of dimethylsulfoxide (DMSO), followed by 0.592 g (1.69 mmol) ofCompound III, prepared according to the general method described inExample 3, 3.727 g (16.90 mmol) of[3-(methacryloylamino)propyl]trimethylammonium chloride (MAPTAC),delivered as 7.08 ml of a 50% aqueous solution, 0.169 ml (1.12 mmol) ofTEMED and 0.333 g (2.03 mmol) of AIBN. The solution was deoxygenatedwith a helium sparge for 4 minutes, followed by an argon sparge for anadditional 4 minutes. The sealed vessel was then heated overnight at 55°C. to complete the polymerization. The DMSO solution was diluted withwater and dialyzed against deionized water using 12,000-14,000 molecularweight cutoff tubing. Lyophilization of the resulting solution gave14.21 g of a white solid. NMR analysis (D₂O) confirmed the presence ofthe methyl groups on the quaternary ammonium groups at 3.10 ppm and thephotogroup load was determined to be 0.101 mmol BBA/g of polymer. TheCompound III constituted 1 mole % of the polymerizable monomer in thisreaction to give Compound X-A.

The above procedure was used to prepare a polymer having 2 mole % ofCompound III. Acrylamide, 10.237 g (0.144 mol), was dissolved in 145 mlof DMSO, followed by 1.148 g (3.277 mmol) of Compound III, preparedaccording to the general method described in Example 3, 3.807 g (17.24mmol) of MAPTAC, delivered as 7.23 ml of a 50% aqueous solution, 0.164ml (1.09 mmol) of TEMED and 0.322 g (1.96 mmol) of AIBN. Workup asdescribed above gave 12.54 g of product (Compound X-B) with a photogroupload of 0.176 mmol BBA/g of polymer.

Example 11 Preparation of Copolymer of Acrylamide, BBA-APMA, MA-EAC-NOS,and [3-(Methacryloylamino)propyl]trimethylammonium Chloride (RandomPhoto PA-PolyNOS-Poly Quat) (Compound XI)

A photoactivatable copolymer of the present invention was prepared inthe following manner. The water in the commercially available 50%aqueous MAPTAC was removed by azeotropic distillation with chloroform.The aqueous MAPTAC solution, 20 ml containing 10.88 g of MAPTAC, wasdiluted with 20 ml of DMSO and 100 ml of chloroform. This mixture wasrefluxed into a heavier-than-water liquid-liquid extractor containinganhydrous sodium sulfate for a total of 80 minutes. A slow flow of airwas maintained during the reflux to inhibit polymerization of themonomer. At the end of the reflux, the excess chloroform was removedunder reduced pressure to leave a DMSO solution of MAPTAC at anapproximate concentration of 352 mg/ml.

Acrylamide, 1.7 g (23.90 mmol), was dissolved in 57.7 ml ofdimethylsulfoxide (DMSO), followed by 0.215 g (0.614 mmol) of CompoundIII, prepared according to the general 1~ method described in Example 3,1.93 ml (0.677 g, 3.067 mmol) of the above MAPTAC/DMSO solution, 0.91 g(3.068 mmol) of Compound V, prepared according to the general methoddescribed in Example 5, and 0.060 g (0.365 mmol) of AIBN. The solutionwas deoxygenated with a helium sparge for 4 minutes, followed by anargon sparge for an additional 4 minutes. The sealed vessel was thenheated overnight at 55° C. to complete the polymerization. The polymerwas isolated by pouring the reaction mixture into 600 ml of diethylether. The solids were separated by centrifuging and the product waswashed with 200 ml of diethyl ether and 200 ml of chloroform.Evaporation of solvent under vacuum gave 3.278 g of product with aphotoload of 0.185 mmol BBA/g of polymer.

Example 12 Copolymer of Acrylamide and MAL-EAC-NOS usingN-(2-Mercaptoethyl)-3,5-bis(4-benzoylbenzyloxy)benzamide (End-pointDiphoto PA-PolyNOS) (Compound XII)

A photoactivatable copolymer of the present invention was prepared inthe following manner. Acrylamide, 3.16 g (44.5 mrol), was dissolved in45.0 ml of tetrahydrofuran, followed by 0.164 g (1 mmol) of AIBN, 0.045ml (0.30 mmol) of TEMED, 0.301 g (0.5 mmol) of Compound VII, preparedaccording to the general method in Example 7, and 1.539 g (5 mmol) ofCompound IV, prepared according to the general method described inExample 4. The solution was deoxygenated with a helium sparge for 4minutes, followed by an argon sparge for an additional 4 minutes. Thesealed vessel was then heated overnight at 55° C. to complete thepolymerization. The precipitated polymer was isolated by filtration andwas washed with chloroform. The final product was dried in a vacuum ovento provide 4.727 g of polymer having a photogroup load of 0.011 mmolBBA/g of polymer.

Example 13 Copolymer of N-[3-(Dimethylamino)propyl]methacrylamide andBBA-APMA (Random Photo Poly Tertiary Amine) (Compound XIII)

A photoactivatable copolymer of the present invention was prepared inthe following manner. N-[3-(Dimethylamino)propyl]methacrylamide, 33.93 g(0.2 mol), was dissolved in 273 ml of DMSO, followed by 16.6 ml ofconcentrated HCl and 6.071 g (17.3 mmol) of Compound III, preparedaccording to the general method described in Example 3. Finally, 0.29 ml(1.93 nmmol) of TEMED, 0.426 g (2.6 mmol) of AIBN, and 100 ml of waterwere added to the reaction mixture. The solution was deoxygenated with ahelium sparge for 10 minutes and the head space was then filled withargon. The sealed vessel was heated overnight at 55° C. to complete thepolymerization. The product was then dialyzed against deionized waterfor several days using 12,000-14,000 MWCO tubing. The product wasfiltered following dialysis to remove any solids and was lyophilized togive 47.27 g of a solid product. The polymer was determined to have aphotoload of 0.33 mmol BBA/g of polymer.

Example 14 Preparation of N-succinimidyl 5-oxo-6-aza-8-nonenoate(Allyl-GLU-NOS) (Compound XIV)

A functional monomer was prepared in the following manner, and was usedin Example 15 to introduce activated ester groups on the backbone of thepolymer. Glutaric anhydride, 20 g (0.175 mole),was dissolved in 100 mlchloroform. The glutaric anhydride solution was cooled to <10° C usingan ice bath. Allyl amine, 10 g (0.177 mole), was dissolved in 50 mlchloroform and added to the cooled solution of glutaric anhydride withstirring. The addition rate of allyl amine was adjusted to keep thereaction temperature <10° C. After the allyl amine addition wascompleted, the reaction solution was allowed to come to room temperaturewhile stirring overnight. After removing the solvent, the5-oxo-6-aza-8-nonenoic acid isolated amounted to 31.4g (105% crude) witha dual DSC melting point of 35.1° C and 44.9° C. NMR analysis at 300 MHzwas consistent with the desired product: 1H NMR (CDCl₃) amide proton6.19 (b, 1H), vinyl protons 5.13, 5.81 (m, 3H), methylene adjacent toamide N 3.85 (m, 2H), methylenes adjacent to carbonyls 2.29, 2.39 (t,4H), and central methylene 1.9. (m, 2H).

The 5-oxo-6-aza-8-nonenoic acid, 20.54 g (0.12 mole),N-hydroxysuccinimide (NHS), 15.19 g (0.13 mole), and 204 ml dioxane wereplaced in a 1 L 3-necked round bottom flask equipped with an overheadstirrer and an addition funnel. Dicyclohexylcarbodiimide (“DCC”), 29.7 g(0.144 mole), was dissolved in 50 ml dioxane and placed in the additionfunnel. The DCC solution was added with stirring to the acid/NHSsolution over 20 minutes, and the resulting mixture was allowed to stirat room temperature overnight. The reaction mixture was filtered on aBüchner finnel to remove dicyclohexylurea (DCU). The solid was washedwith 2×100 ml dioxane. The solvent was evaporated to give 41.37 gresidue, which was washed with 4×75 ml hexane. After the solvents wereremoved, the yield of crude NOS ester was 41.19 g. One recrystallizationof the crude NOS product from toluene gave a 60% yield with a DSCmelting point of 90.1° C. NMR analysis at 300 MHz was consistent withthe desired product: ¹H NMR (CDCl₃) amide proton 6.02 (b, 1H), vinylprotons 5.13, 5.80 (m, 3H), methylene adjacent to amide N 3.88 (m, 2H),succinimidyl protons 2.83 (s, 4H), methylenes adjacent to carbonyls2.31, 2.68 (t, 4H), and central methylene 2.08 (m, 2H). The finalcompound was stored for use in the synthesis of photoactivatablepolymers as described in Example 15.

Example 15 Preparation of Copolymer of Vinylpyrrolidinone, BBA-APMA, andAllyl-GLU-NOS (Random Photo PVP-PolyNOS)(Compound XV)

A photoactivatable copolymer of the present invention was prepared inthe following manner. Vinylpyrrolidinone, 4.30 g (38.73 mmol), wasdissolved in 5.2 ml of DMSO along with 0.14 g (0.41 mmol) of CompoundIII, prepared according to the general method described in Example 3,0.55 g (2.06 mmol) Compound XIV, prepared according to the generalmethod described in Example 14, by combining 0.08 g (0.49 mmol) of AIBNand 0.005 ml (0.033 mmol) of TEMED. The solution was deoxygenated with ahelium sparge for 3 minutes. The head space was replaced with argon, andthe vessel was sealed for an overnight heating at 55° C. The viscoussolution was diluted with 15 ml chloroform, and then precipitated bypouring into 200 ml diethyl ether. The precipitate was dissolved in 15ml chloroform, and precipitated a second time in 200 ml ether. Theproduct was dried in a vacuum oven at 30° C. to give 4.79 g of a whitesolid. NMR analysis (CDCl₃) confirmed the presence of the NOS group at2.81 ppm and the photogroup load was determined to be 1.1 mmol BBA/g ofpolymer. The Allyl-GLU-NOS composed 5.0 mole % of the polymerizablemonomers in this reaction to give Compound XV.

Example 16 Comparison of Random Photo PA-PolyNOS (Compound IX-C) withRandom Photo PA-PolyNOS-PolyQuat (Compound XI) on Polystyrene (PS)Microwell Plates

Compound IX-C and Compound XI were separately dissolved in deionizedwater at 5 mg/ml. The PS plates (PS, Medium Bind, Coming Costar,Cambridge, MA) containing 100 μl of Compound IX-C and Compound XI inseparate wells were illuminated with a Dymax lamp (model no. PC-2, DymaxCorporation, Torrington, Conn.) which contained a Heraeus bulb (W. C.Heraeus GmbH, Hanau, Federal Republic of Germany). The illuminationduration was for 1.5 minutes at a intensity of 1-2mW/cm² in thewavelength range of 330-340 rm. The coating solution was then discardedand the wells were air dried for two hours. The plates were thenilluminated for an additional one minute. The coated plates were usedimmediately to immobilize oligonucleotides stored in a sealed pouch forup to 2 months.

The 50 base oligomer (-mer) capture probe5′-NH₂-GTCTGAGTCGGAGCCAGGGCGGCCGCCAACAGCAGGAGCAGCGTGCACGG-3′ (SEQ IDNO:1) (synthesized with a 5′-amino-modifier containing a C-12 spacer) at10 pmoles/well was incubated in PS wells in 50 mM phosphate buffer, pH8.5, 1 mM EDTA at 37° C. for one hour. The hybridization was performedas follows using the complementary5′-Biotin-CCGTGCACGCTGCTCCTGCTGTTGGCGGCCGCCCTGGCTCCGACTC AGAC -3′(SEQ IDNO:3) detection probe or non-complementary5′-Biotin-CGGTGGATGGAGCAGGAGGGGCCC GAGTATTGGGAGCGGGAGACA CAGAA-3′ (SEQID NO:4) oligo, both of which were synthesized with a 5′-biotinmodification.

The plates with immobilized capture probe were washed with phosphatebuffered saline (PBS, 10 mM Na₂PO₄, 150 mM NaCl, pH 7.2) containing0.05% Tween 20 using a Microplate Auto Washer (model EL 403H, Bio-TekInstruments, Winooski, Vt.). The plates were then blocked at 55° C. for30 minutes with hybridization buffer, which consisted of 5X SCC (0.75 MNaCl, 0.075 M citrate, pH 7.0), 0.1% lauroylsarcosine, 1% casein, and0.02% sodium dodecyl sulfate. When the detection probe was hybridized tothe capture probe, 50 fmole of detection probe in 100 μl were added perwell and incubated for one hour at 55° C. The plates were then washedwith 2X SSC containing 0.1% sodium dodecyl sulfate for 5 minutes at 55°C. The bound detection probe was assayed by adding 100 μl of a conjugateof streptavidin and horseradish peroxidase (SA-HRP, Pierce, Rockford,Ill.) at 0.5 μg/ml and incubating for 30 minutes at 37° C. The plateswere then washed with PBS/Tween, followed by the addition of peroxidasesubstrate (H₂O₂ and tetramethylbenzidine, Kirkegard and PerryLaboratories, Gaithersburg, Md.) and measurement at 655 nm on amicrowell plate reader (model 3550, Bio-Rad Labs, Cambridge, Mass.). Theplates were read at 10 minutes.

The results listed in Table 1 indicate that microwell plates coated withCompound IX-C did not effectively immobilize amine-derivatized captureprobes. However, by comparison Compound XI, as a coating, providedsignificant binding and good hybridization signals. Compound IX-Creagent most likely passivated the surfaces and prevented theassociation of capture oligos. In contrast when Compound XI was used,the oligonucleotide was attracted to the surface by ionic interactionswhere it could then be covalently bonded with the NOS groups.

TABLE 1 Hybridization Signals (A₆₅₅) from PS Microwell Plates Coatedwith Compound IX-C and Compound XI. Compound IX-C Compound XIComplementary 0.187 ± 0.031 1.666 ± 0.064 Detection ProbeNon-complementary 0.127 ± 0.016 0.174 ± 0.005 Detection Probe

Example 17 Coating of Various Microwell Plates with a Mixture of RandomPhoto PA-PolyNOS (Compound IX-B) and Random Photo PA-PolyQuat (CompoundX-B)

A coating solution containing a mixture of 5 mg/ml of Compound IX-B and0.5 mg/ml of Compound X-B was prepared in deionized water. This mixturewas used to treat polypropylene (PP, Coming Costar, Cambridge, Mass.),PS, polycarbonate (PC, Coming Costar, Cambridge, Mass.) and polyvinylchloride (PVC, Dynatech, Chantilly, Va.) multiwells as described inExample 16. A 30-mer capture oligonucleotide5′-NH₂-GTCTGAGTCGGAGCCAGGGCGGCCGCCAAC-3′(SEQ ID NO:2), (synthesized with a5′-amino-modifier containing a C-12 spacer) at 0.03, 0.1, 0.3, 1, 3, or10 pmole/well was incubated at 4° C. overnight. The hybridization wasperformed as previously described in Example 16 using complementary SEQID NO:3 detection oligonucleotide or non-complementary SEQ ID NO:4oligo. Since PP plates are not optically transparent, the contents ofeach well were transferred to PS wells after a 20 minute incubation withthe chromogenic substrate. The hybridization signals were measured inthe PS plates. The other plates were read without transferring at 10minutes. Signal levels are only comparable within the same substrategroup due to the different geometries of microwell plates made fromdifferent materials. Table 2 lists the hybridization signals and showsthe relationship between the intensity of the hybridization signals andthe amount of capture probe applied to various microwell plates coatedwith a mixture of Compound IX-B and Compound X-B. On PP and PVC plates,adsorption of probes was very low and the coatings with the polymericreagents improved the signals dramatically. The signal increased withincreasing capture probe added to the coated wells, but leveled off atapproximately 3 pmole/well capture. The plateau in the amount of signalgenerated was not due to a saturating level of hybridization, but ratherto the limits of the color change reaction in the calorimetric assay.

Oligonucleotide derivatives adsorb efficiently onto uncoated PS and PCmicrowell plates and result in specific hybridization signals. Cros etal. (U.S. Pat. No. 5,510,084) also reported that amine-functionalizedoligonucleotides adsorbed satisfactorily onto polystyrene microwellplates by unknown mechanisms. However, there is marked variability inthe amount of adsorption on uncoated PS plates among different lots(Chevier et al. FEMS 10:245, 1995).

TABLE 2 Hybridization Signals (A₆₅₅) From Various Microwell PlateMaterials Coated With a Mixture of Compound IX-B and Compound X-BCapture Oligonucleotide Added (pmole/well) 0.03 0.1 0.3 1 3 10 Comp NCComp NC Comp NC Comp NC Comp NC Comp NC PP Uncoated 0.083 0.082 0.0760.072 0.076 0.074 0.088 0.074 0.070 0.067 0.078 0.073 Coated 0.541 0.0991.070 0.099 1.769 0.091 2.283 0.094 2.582 0.141 2.490 0.320 PVC Uncoated0.074 0.079 0.081 0.075 0.097 0.078 0.137 0.076 0.215 0.081 0.337 0.092Coated 0.423 0.116 0.875 0.110 1.326 0.112 1.583 0.142 1.628 0.186 1.6040.332 PS Uncoated 0.235 0.099 0.435 0.091 0.827 0.090 1.205 0.093 1.3800.093 1.404 0.136 Coated 0.435 0.121 0.801 0.105 1.177 0.116 1.401 0.1321.470 0.132 1.487 0.302 PC Uncoated 0.676 0.248 1.364 0.244 2.103 0.2562.701 0.266 2.745 0.295 2.930 0.388 Coated 1.034 0.327 1.602 0.306 2.1360.295 2.218 0.287 2.380 0.342 2.500 0.572 Comp.: Complementary detectionprobe was added for hybridization. NC: Non-complementary detection probewas added for hybridization.

Example 18 Evaluation of End-point Diphoto PA-polyNOS (Compound XII) andRandom Photo PA-PolyQuat (Compound X-B) on PP and PVC Microwell Plates

A coating solution containing a mixture of 5 mg/ml of Compound XII and0.5 mg/ml of Compound X-B was prepared with deionized water. Thismixture of the two reagents was used to coat PP and PVC microwell platesunder conditions comparable to those described in Example 16. The 30-merSEQ ID NO:2 capture oligonucleotide at 0.03, 0.1, 0.3, 1, 3, or 10pmole/well in 0.1 ml was incubated at 4° C. overnight. The hybridizationwas performed as described in Example 16 using complementary SEQ ID NO:3detection oligonucleotide or non-complementary SEQ ID NO: 4oligonucleotide. The hybridization signals listed in Table 3 demonstratethe relationship between the intensity of the hybridization signals andthe amount of capture probe applied to PP and PVC microwell platescoated with a mixture of Compound XII and Compound X-B. The signalincreased with increasing capture oligonucleotides added to the coatedwells, but leveled off at approximately 1 pmole/well. Thesignal-to-noise ratio (from complementary vs. non-complementarydetection probes) was as high as 26 and 11 for coated PP and PVCsurfaces, respectively.

TABLE 3 Hybridization Signals (A₆₅₅) From PP and PVC Plates Coated WithMixture of Compound XII and Compound X-B. PP Microwell plates PVCMicrowell plates pmole/well Comp. Comp. Capture Added DetectionNon-comp. Detection Non-comp.  0.03 0.153 ± 0.070 ± 0.289 ± 0.094 ±0.008 0.007 0.029 0.020 0.1 0.537 ± 0.075 ± 0.759 ± 0.104 ± 0.042 0.0090.054 0.014 0.3 1.206 ± 0.080 ± 1.262 ± 0.117 ± 0.106 0.003 0.023 0.011 1 2.157 ± 0.081 ± 1.520 ± 0.189 ± 0.142 0.003 0.044 0.064  3 2.624 ±0.108 ± 1.571 ± 0.179 ± 0.162 0.012 0.031 0.016 10 2.921 ± 0.200 ± 1.625± 0.286 ± 0.026 0.018 0.040 0.021

Example 19 Sequential Coating with Random Photo PA-PolyQuat (CompoundX-B) and BBA-AUD-NOS (Compound VIII)

Compound X-B at 0.1 mg/ml in deionized water was incubated in PP and PVCwells for 20 minutes. The plates were illuminated as previouslydescribed in Example 16 with the solution in the wells for 1.5 minutes.The solution was discarded and the wells were dried. Compound VIII at0.5 mg/ml in isopropyl alcohol (IPA) was incubated in the Compound X-Bcoated wells for 5 minutes. The solution was then removed, the platedried and illuminated as described in Example 16 for one minute afterthe wells were dried. The 30-mer SEQ ID NO:2 capture oligonucleotide at0.03, 0.1, 0.3, 1, 3, or 10 pmole/well in 0.1 ml was incubated at 4° C.overnight. The hybridization was performed as described in Example 16using complementary (SEQ ID NO:3) detection oligonucleotide ornon-complementary SEQ ID NO:4 oligo. Table 4 contains the hybridizationsignals and shows the relationship between the intensity of thehybridization signals and the amount of capture probe applied to PP andPVC microwell plates coated with Compound X-B followed by Compound VIIIcoating. The signal increased with increasing capture probe added to thecoated wells, but leveled off at approximately 1 pmole/well captureoligo. The signals were up to 29- and 11- fold higher for coated PP andPVC surfaces, respectively, as compared to the uncoated controls.

TABLE 4 Hybridization Signals (A₆₅₅) From PP and PVC Microwell PlatesCoated With Compound X-B Followed by Compound VIII Coating. pmole/wellPP Microwell plates PVC Microwell plates Capture Added Uncoated CoatedUncoated Coated  0.03 0.083 ± 0.157 ± 0.074 ± 0.244 ± 0.003 0.004 0.0040.014 0.1 0.076 ± 0.544 ± 0.081 ± 0.694 ± 0.003 0.006 0.005 0.065 0.30.076 ± 1.095 ± 0.097 ± 1.113 ± 0.006 0.015 0.010 0.033  1 0.088 ± 1.676± 0.137 ± 1.304 ± 0.006 0.030 0.016 0.027  3 0.070 ± 1.865 ± 0.215 ±1.237 ± 0.010 0.057 0.023 0.013 10 0.078 ± 2.274 ± 0.337 ± 1.182 ± 0.0090.005 0.024 0.041

Example 20 Comparision of Random Photo PA-PolyQuat (Compound X-A) with aMixture of Random Photo PA-PolyNOS (Compound IX-A) and Random PhotoPA-PolyQuat (Compound X-A)

Compound X-A at 0.5 or 0.1 mg/ml was incubated in PP microwell platesfor 10 minutes. The plates were then illuminated as described in Example16. A coating solution containing a mixture of Compound IX-A andCompound X-A was prepared at two ratios, 5/0.5 mg/ml and 0.5/0.1 mg/mlof Compound IX-A/Compound X-A in deionized water to coat PP microwellplates. The solution was incubated in the wells for 10 minutes and thewells were illuminated as described in Example 16. The 30-mer SEQ IDNO:2 capture oligonucleotide at 1 pmole/well was incubated in each wellat 37° C. for one hour. The hybridization was done as described inExample 16 using complementary SEQ ID NO:3 detection oligonucleotide ornon-complementary SEQ ID NO:4 oligo. The results listed in Table 5indicate that the coating containing the combination of Compound IX-Aand Compound X-A gave higher signals as compared to those from CompoundX-A coating alone.

TABLE 5 Hybridization Signals (A₆₅₅) From Compound X-A Coated PPMicrowell Plates. Ratio of Compound IX- A/Compound X-A (mg/ml) Comp.Detection Non-comp. Detection 5/0.5 1.436 ± 0.056 0.077 ± 0.001 0/0.50.454 ± 0.149 0.052 ± 0.006 0.5/0.1   1.346 ± 0.044 0.062 ± 0.003 0/0.10.192 ± 0.082 0.055 ± 0.002

Example 21 Comparision of Non-modified Oligonucleotide vs.Amine-Modified Oligonucleotide on Random Photo PA-PolyNOS (CompoundIX-B) and Random Photo PA-PolyQuat (Compound X-B) on Coated MicrowellPlates

A coating solution containing a mixture of Compound IX-B (5 mg/ml) andCompound X-B (0.5 mg/ml) was prepared in deionized water to coat PP, PSand PVC microwell plates. The solution was incubated for approximately10 minutes and illuminated as described in Example 16. The 30-mercapture 5′-NH₂-TTCTGTGTCTCC CGCTCCCAATACTCGGGC-3′(SEQ ID NO:5)oligonucleotide at 1 pmole/well was coupled to the wells in 50 MMphosphate buffer, pH 8.5, 1 mM EDTA at 4° C. overnight. Thehybridization was performed as described in Example 16 usingcomplementary detection oligonucleotide SEQ ID NO:4 or non-complementaryoligonucleotide (SEQ ID NO:3). To determine the effect of theamine-functionality of the capture oligo, a non-modified 30-mer captureprobe 5′-TTCTGTGTCTCC CGCTCCCAATACTCGGGC-3′(SEQ ID NO:6) (with no amine)was also added to the coated surfaces and tested. The results shown inTable 6 indicate that when an oligonucleotide without the 5′-aminemodification was used as the capture probe on Compound IX-B/Compound X-Bcoated surfaces, the hybridization signal was less than 30% of that withamine modification.

TABLE 6 Signals (A₆₅₅) Generated From Hybridization Reactions WithEither SEQ ID NO:5 or SEQ ID NO:6 Oligonucleotides on CompoundIX-B/Compound X-B Coated Microwell Plates. No Capture Added Non-modifiedCapture Amine-modified Capture Comp. Non-comp. Comp. Non-comp. Comp.Non-comp. Detection Detection Detection Detection Detection Detection PPUncoated 0.032 ± 0.001 0.036 ± 0.004 0.033 ± 0.001 0.036 ± 0.001 0.037 ±0.005 0.033 ± 0.001 Coated 0.038 ± 0.002 0.040 ± 0.001 0.555 ± 0.0410.044 ± 0.001 1.915 ± 0.029 0.066 ± 0.003 PVC Uncoated 0.248 ± 0.0490.176 ± 0.008 0.259 ± 0.049 0.128 ± 0.013 0.404 ± 0.100 0.118 ± 0.025Coated 0.115 ± 0.027 0.090 ± 0.014 0.379 ± 0.028 0.091 ± 0.014 1.319 ±0.027 0.101 ± 0.017 PS Uncoated 0.084 ± 0.013 0.089 ± 0.014 0.668 ±0.047 0.085 ± 0.023 1.269 ± 0.034 0.106 ± 0.024 Coated 0.080 ± 0.0060.081 ± 0.023 0.364 ± 0.010 0.089 ± 0.015 1.437 ± 0.012 0.098 ± 0.005

Example 22 Oligonucleotide Loading Densities on Microwell Plates Coatedwith Random Photo PA-PolyNOS (Compound IX-A) and Random PhotoPA-PolyQuat (Compound X-A)

Radiolabeled assays were performed to determine oligonucleotide loadingdensities and to verify results from the colorimetric assay system. Inthis Example, combination coatings of Compound IX-A and Compound X-Awere performed on PVC wells as described in Example 16. The SEQ ID NO:2and SEQ ID NO:5 30-mer capture oligonucleotides were immobilized oncoated wells. A radiolabeled SEQ ID NO:2 probe was used to determine theloading density of immobilized capture oligonucleotides on the wellsurface. A radiolabeled SEQ ID NO:3 detection probe, which wascomplementary to SEQ ID NO:2, but not to SEQ ID NO:5, was used tomeasure hybridization reactions of the immobilized capture probes.Oligonucleotides SEQ ID NO:2 and SEQ ID NO:3 were radiolabeled at the3′-end using terminal transferase (Boehringer Mannheim, Indianapolis,Ind.) and α-³²P-ddATP (3000 Ci/mmole, Amersham, Arlington Heights, Ill.)according to the manufacturer's specifications. ³²P-labeled SEQ ID NO:2and unlabeled SEQ ID NO:2 and SEQ ID NO:5 capture probes were incubatedin coated wells at 50 pmole/well for 2.25 hours at room temperature. Theplates were washed and blocked as in Example 16.

The wells with the unlabeled capture probes were hybridized with the³²P-labeled SEQ ID NO:3 detection probe in hybridization buffer for 1hour at 55° C. Wells containing the ³²P-labeled capture probe wereincubated in hybridization buffer without the SEQ ID NO:3 probe. Afterwashing three times with 2X SSC containing 0.1% SDS for 5 minutes at 550C and three times with PBS/0.05% Tween, the plates were cut intoindividual wells and dissolved in tetrahydrofuran. The amount ofradioactivity in each well was measured by scintillation counting inAquasol-2 Fluor (DuPont NEN, Boston, Mass.). The results in Table 7 showthat both Compound IX-A and Compound X-A were required to give goodimmobilization of capture probe. Also, increasing the concentrations ofCompound IX-A and Compound X-A increased the amount of the captureoligonucleotide immobilized. At the highest concentrations tested, thesignal to noise ratio was greater than 3000 to 1.

TABLE 7 Densities of Immobilized Capture Oligonucleotide and Hybridized³²P-Detection Oligo. Hybridized Hybridized Mixture of Coating ReagentsImmobilized comp. non-comp. Compound Compound capture detectiondetection IX-A (mg/ml) X-A (mg/ml) fmole/well fmole/well fmole/well 0 041.3 2.3 0.6 0 0.05 37.5 10.9 0.7 0.55 0 32.6 5.4 0.6 1 0.1 344.1 308.826.4 0.1 0.1 285.7 222.2 55.7 1 0.001 52.8 26.2 0.6 0.1 0.001 73.5 20.813.1 1.19 0.05 280.4 256.9 1.1 0.55 0.12 401.9 379.1 0.7 0.55 0.05 338.0315.1 1.6 2 0.5 1633.4 1108.4 0.3

Example 23 Comparison between Random Photo-Polytertiary Amine (CompoundXIII), Random Photo-PA-PolyNOS (Compound IX-A) and a Mixture of RandomPhoto PA-PolyNOS (Compound IX-A) and Random Photo-Polytertiary Amine(Compound XIII)

Compound XIII at 0.02 mg/ml in deionized water was incubated in PPmicrowell plates for 10 minutes. The wells were illuminated as describedin Example 16. Compound IX-A was coated on PP wells at 2 mg/ml indeionized water as described for Compound XIII. A coating solutioncontaining a mixture of 2 mg/ml Compound IX-A and 0.02 mg/ml CompoundXIII in deionized water was prepared and coated as described forCompound XIII. The 30-mer SEQ ID NO:2 capture oligonucleotide at 5pmole/well was incubated in each well at 37° C. for one hour. Thehybridization was done as described in Example 16 using complementarySEQ ID NO:3 detection oligonucleotide and non-complementary SEQ ID NO:4oligonucleotide. The contents of each well were transferred to PS wellsafter a 10 minute incubation with the peroxidase substrate. The resultslisted in Table 8 indicate that the combination of Compound IX-A andCompound XIII gave higher signals compared to those from Compound IX-Aor Compound XIII coating alone.

TABLE 8 Hybridization Signals (A₆₅₅) From Coated PP Microwell Plates.Coating Comp. Detection Non-comp. Detection Compound IX-A 0.057 ± 0.0010.052 ± 0.006 Compound XIII 0.746 ± 0.042 0.081 ± 0.009 CompoundIX-A/Compound 1.195 ± 0.046 0.078 ± 0.014 XIII Mixture

Example 24 Nucleic Acid Sequence Immobilization on an Amine DerivatizedSurface

A copolymer of the present invention is prepared in the followingmanner. Acrylamide, 5.686 g (80.0 mmol), is dissolved in 100 ml of DMSO,followed by the addition of 3.083 g (10.0 mmol) of Compound IV, preparedaccording to the general method described in Example 4, and 2.207 g(10.0 mmol) of MAPTAC, delivered as a dry DMSO solution preparedaccording to the general method described in Example 11. TEMED, 0.134 ml(0.89 mmol), and AIBN, 0.197 g (1.20 mmol), are added to the mixture andthe system is deoxygenated with a helium sparge for 5 minutes, followedby an argon sparge for an additional 5 minutes. The sealed vessel isheated at 55° C. to complete the polymerization. The polymer is isolatedby pouring the reaction mixture into 800 ml of diethyl ether andcentrifuging to separate the solids. The product is washed with 200 mlof diethyl ether, followed by 200 ml of chloroform. The polymer is driedunder vacuum to remove remaining solvent.

A polymer surface is derivatized by plasma treatment using a 3:1 mixtureof methane and ammonia gases. (See, e.g., the general method describedin U.S. Pat. No. 5,643,580). A mixture of methane (490 SCCM) and ammonia(161 SCCM) are introduced into the plasma chamber along with the polymerpart to be coated. The gases are maintained at a pressure of 0.2-0.3torr and a 300-500 watt glow discharge is established within thechamber. The sample is treated for a total of 3-5 minutes under theseconditions. Formation of an amine derivatized surface is verified by areduction in the water contact angle compared to the uncoated surface.

The amine derivatized surface is incubated for 10 minutes at roomtemperature with a 10 mg/ml solution of the above polymer in a 50 mMphosphate buffer, pH 8.5. Following this reaction time, the coatingsolution is removed and the surface is washed thoroughly with deionizedwater and dried thoroughly. Immobilization of oligomer capture probe andhybridization is performed as described in Example 16.

Example 25 Immobilization and Hybridization of Oligonucleotides onPhoto-Polymeric NOS Coated Glass Slides—Comparison of coatings with andwith out Photo PA PolyQuat (Compound X-A)

Soda lime glass microscope slides (Erie Scientific, Portsmouth, NewHampshire) were silane treated by dipping in a mixture ofp-tolyldimethylchlorosilane (T-Silane) and N-decyldimethylchlorosilane(D-Silane, United Chemical Technologies, Bristol, Pennsylvania), 1% eachin acetone, for 1 minute. After air drying, the slides were cured in anoven at 120° C. for one hour. The slides were then washed with acetonefollowed by DI water dipping. The slides were further dried in oven for5-10 minutes.

Compound IX-A, IX-D, and XV at various concentrations and with orwithout Compound X-A, were sprayed onto the silane treated slide, whichwere then illuminated using a Dymax lamp (25 mjoule/cm² as measured at335 nm with a 10 nm band pass filter on an International Lightradiometer) while wet, washed with water, and dried. Oligonucleotideswere printed on the slides using an X, Y, Z motion controller toposition a 0.006″ id blunt end needle filled with oligonucleotidesolution. Two oligonucleotides were immobilized to the prepared slides.One containing an amine on the 3′ end and Cy3 fluorescent tag (Amersham,Arlington Heights, Ill.) on the 5′ end, 5′Cy3-GTCTGAGTCGGAGCCAGGGCGGCCGCCAAC-NH2-3′(SEQ ID NO:7) (amino modifierhas a C12 spacer) and the other containing an amine on the 5′ end,5′-NH2-TTCTGTGTCTCCCGCTCCCAATACTCGGGC-3′(SEQ ID NO:5) ) (amino modifierhas a C12 spacer). They were printed at a concentration of 8 pmole/ptlin 50 mM sodium phosphate pH 8.5 containing 10% sodium sulfate and 1 mMEDTA. Slides were placed overnight on a rack in a sealed container withsaturated sodium chloride to maintain a relative humidity of 75%. Slidesprinted with (SEQ ID NO:7) were then washed for 5 minutes in PBS/0.05%Tween-20, for 90 minutes in blocking buffer (0.2 M Tris with 10 mMethanolamine) at 500 C, and for 2 hours in wash buffer (5X SSC, 0.1%N-lauryl sarcosine, and 0.1% sodium dodecyl sulfate). Slides were washedtwice with water and spun in a centrifuge to dry. They were than scannedusing a General Scanning Scan-Array 3000 fluorescence scanner(Watertown, Massachusetts) and the average intensities of the resultingspots were measured. Slides printed with (SEQ ID NO:5) were washed for 5minutes in PBS/0.05% Tween-20 and for 30 minutes in blocking buffer (0.2M Tris with 10 mM ethanolamine) at 50° C. The slides were finally washedwith water and dried in a centrifuge.

Fluorescently labeled complementary oligonucleotide,5′-Cy3-CGGTGGATGGAGCAGGAGGGGCCCGAGTATTGGGAGCGGGAGACACAGAA-3′(SEQ IDNO:8), was hybridized to the slides by placing 10 IIl of hybridizationsolution (4X SSC, 0.1% N-laurylsarcosine, 2 mg/ml tRNA) on the slide andplacing a cover slip on top. The slides were then kept at 50° C. highhumidity (75%) to prevent drying out of the hybridization solution.Slides were then rinsed with 4X SSC, 2X SSC preheated to 50° C. for 2minutes, 2X SSC for 2 minutes, and then twice into 0.1X SSC for 2minutes each. Slides were spun dry in a centrifuge. They were thenscanned using a General Scanning fluorescence scanner. Averageintensities of the resulting spots and background levels were measured.The results listed in Table 9 show that the coatings without compoundX-A immobilize slightly less oligonucleotide but hybridization of afluorescent oligonucleotide results in slightly higher signal. Theresulting background is less on coatings which do not contain compoundX-A. It also shows that polymers containing PVP backbone compound (i.e.Compound XV) are effective at immobilizing DNA and give goodhybridization results.

TABLE 9 Immobilization and Hybridization of Oligonucleotides to GlassMicroscope Slides. immobilized hybridization Compound Poly-NOS Cmpd X-ASEQ ID NO:7 SEQ ID NO:8 % BBA  % NOS conc g/l conc g/l signal¹ signal²bkg S/N Compound IX-A 1.25 0 39151 38512 45 856 Compound IX-A 1 0.2542598 35674 88 405 Compound IX-A 2.5 0 35153 31061 34 914 Compound IX-A2 0.5 44233 24735 75 332 Compound IX-D 1.25 0 30655 41669 45 926Compound IX-D 1 0.25 38594 34300 99 346 Compound IX-D 2.5 0 41226 4897667 736 Compound IX-D 2 0.5 46444 22743 123  185 Compound XV 1.25 0 2822850248 34 1478  Compound XV 1 0.25 31544 47321 97 488 ¹Laser power set at60% and photomultiplier tube set at 60% ²Laser power set at 80% andphotomultiplier tube set at 80%

Example 26 Hybridization of Immobilized PCR products on Coated GlassSlides with OligonucleotideDetection Probe. Comparison between RandomPhoto-PA-PolyNOS (Compound IX-A) and a Mixture of RandomPhoto-PA-PolyNOS (Compound IX-A) and Random Photo-PA-PolyQuat (CompoundX-A).

Glass slides were coated with organosilane as described in Example 25.Compound IX-A at 1.25 mg/ml in water or a mixture of 1 mg/ml CompoundIX-A and 0.25 mg/ml Compound X-A in water was coated onto silane treatedglass slides as described in Example 25.

PCR products from β-galactosidase gene were custom prepared by ATGLaboratories, Inc. (Eden Prairie). Primer with 5′-amine modification onthe sense strand and unmodified primer on the anti-sense strand wereused to prepare double-stranded- PCR products at 0.5 and 1 kilobase (kb)pair length. The control DNAs without amine were also made. The DNAs atconcentration 0.2 μg/μl in 80 mM sodium phosphate buffer, pH 8.5, and 8%sodium sulfate were printed on the activated slides using microarrayingspotting pins from TeleChem International (San Jose, Calif.). Thecoupling was allow to proceed in a sealed container with 75% humidityovernight at room temperature.

To evaluate the signals from immobilized PCR products on microarrays,the slides were placed in boiling water for 2 minutes to denaturedouble-stranded DNA and to remove the non-attached strand. The slideswere then incubated with 50 mM ethanolamine in 0.1 M Tris buffer, pH 9at 50° C. for 15 minutes to block residual reactive groups on thesurfaces. The slides were then incubated with pre-hybridization solutionunder glass cover slips at 50° C. for 15 minutes to decrease thenon-specific backgrounds. The pre-hybridization solution contained 5XSSC, 5X Denhardt's solution (0.1 mg/ml each of bovine serum albumin,Ficoll and PVP), 0.1 mg/ml salmon sperm DNA and 0.1% SDS. Thehybridization was then performed with 20 fmole/μl of a fluorescentcomplementary detection oligo, 5′-Cy3-ACGCCGA GTTAACGCCATCA (SEQ IDNO9), in the pre-hybridization solution overnight at 45° C. Slides werethen washed and the hybridization signals scanned as described inExample 25.

The results listed in Table 10 indicate that the glass slides coatedwith Compound IX-A and mixture of Compound IX-A/X-A had comparablesignals. Amine-containing PCR product had at least 30-fold higherhybridization signals than non-modified DNA. The low level of signalswith unmodified DNA was probably due to side reactions between amines onthe heterocyclic bases to the activated surfaces.

TABLE 10 Hybridization Signals With Immobilized 0.5 Kb DNA And aComplementary Detection Oligonucleotide SEQ ID NO:9 on CompoundIX-A/Compound X-A Coated Glass Slides. Amine-primer Non-modified primerCoating PCR product PCR product Compound IX-A 10,385 ± 2,379 341 ± 61Compound IX-A and 16,858 ± 4,008 341 ± 79 Compound X-A Mixture

Example 27 Hybridization of Immobilized PCR products on Coated GlassSlides with Oligonucleotide Detection Probe—Comparison between SurModicsand other Commercial Slides.

PCR products from cDNA clones can be attached to the positively chargedglass surfaces, such as polylysine; DeRisi, et. al., (Science, 278,680-686, 1997), and a covalent approach having aldehyde groups has beenreported by Schena (Schena et.al., Proc. Natl. Acad. Sci. USA, 93,10614-10619). In this example PCR products were attached to thosesurfaces and the hybridization signals were compared with the coatingsfrom this invention. SurModics glass slides were coated with mixture ofCompound IX-A and Compound X-A as described in Example 25. Silylatedglass slides that have reactive aldehyde groups for immobilizingamine-functionalized DNA was manufactured by CEL Associates, Inc.(Houston, Tex.). Polylysine glass slides were purchased from Sigma.

PCR products at 1 kb length from β-galactosidase at 1.5 pmole/μl in 50mM sodium phosphate buffer, pH 8.5, 1 mM EDTA and 3% sodium sulfate wereprinted onto silylated slides, polylysine slides and SurModics coatedslides using 0.006″ id needle as described in Example 25. The SurModicsslides were then incubated in 75% relative humidity chamber for 2 days,denatured by submerging in boiling water bath for 2 minutes, and blockedwith 10 mM ethanolamine, 0.2 M Tris, pH 8.5 for 30 minutes at 50° C. Thesilylated slides were incubated in a humidified incubator for 4 hoursand then reduced with sodium borohydride as suggested by themanufacturer. The polylysine slides were UV crosslinked and then blockedwith succinic anhydride as described in the literature¹. All theprocessed slides were hybridized with 20 finole/μl of complementarydetection oligonucleotide SEQ ID NO:9 in 4X SSC, 2 mg/ml tRNA, 0.1 %lauroylsarcosine at 45° C. overnight. The slides were washed andhybridization signals were scanned as described in Example 25.

The results are shown in the following Table 11. There was no differencein signals between amine-modified versus unmodified DNA on silylated andpolylysine slides. Only SurModics coatings demonstrated that specificattachment was due to having a 5′-amine on the PCR products. Thisprovides evidence of end-point attachment of DNA up to 1 kb withSurModics coatings. Polylysine slides had the highest backgroundprobably due to ionic and/or inon-specific binding of the DNA onto thesurfaces.

TABLE 11 Hybridization Signals With Immobilized 1 Kb DNA and aComplementary Detection Oligonucleotide SEQ ID NO:9 on Coated GlassSlides. Comparison of Compound IX-A/ Compound X-A Coated Slides andCommercial Glass Slides. Non- Amine-primer modified primer Coating PCRproduct PCR product Background Compound IX-A 26,580 ± 3,219 946 ± 185 88 and Compound X-A Mixture Silylated 5,611 ± 2,063 7,050 ± 2,211 114Polylysine 4,3674 ± 2,832  4,3206 ± 4,743  3,075  

Example 28 Immobilization and Hybridization of PCR Products with cDNADetection Probe on Photo-Polymeric NOS Coated Glass Slides.

Two sets of slides were prepared as described in Example 26. Three PCRproduct sequences (designated FI 1, XEF, daf) containing an amine onboth, the forward, the reverse or neither strand (provided by AxysPharmaceuticals, La Jolla, Calif.) were dissolved in printing buffer(80ng/μl), heated at 100° C., cooled on ice, and printed on the slidesusing a Generation II Arrayer (Molecular Dynamics, Sunnyvale,California). After incubation overnight as described in Example 25, theslides were placed in a boiling water bath for 2 minutes, washed twicewith PBS/0.05% tween-20, rinsed twice with water, and put in blockingbuffer for 30 minutes at 50° C. The slides were than rinsed with waterand spun dry. Slides were prehybridized as described in Example 26 andhybridized to a mixture of fluorescently (Cy3) labeled cDNA (provided byAxys Pharmaceuticals) in 50% formamide, 5X SSC, 0.1% SDS, and 0.1 mg/mlsalmon sperm DNA at 42° C. overnight. This mixture containedcomplementary probes to the forward strand of all three PCR producttargets. The Fl I probe was spiked at a 1 to 50,000 mass ratio relativeto the other two sequences. After hybridization, the slides were washedand scanned as described in Example 25. The average intensities of thespots are shown in Table 12. Slides which were hybridized to a cDNAprobe mixture which did not contain the F11 probe showed no signal inthese spots. The results show that both coating types give comparablehybridization results. The coating containing compound X-A had muchhigher background. This was especially true in the area near where thePCR product was printed.

TABLE 12 Immobilization of PCR Products and Hybridization toFluorescently Labeled cDNA on Glass Microscope Slides. Numbers areFluorescent Signal¹. amine on coated with both forward reverse neithercompound IX-A strands strand strand strand 0.85 Kb XEF 2664.5 6125.5759.5 3590.5 1 Kb daf 42921.5 14294 1 Kb F11 588 1859.5 123.5 891.5background = 80 amine on coated with mixture both forward reverseneither compounds IX-A & X-A strands strand strand strand 0.85 Kb XEF3001 12896 779 4119 1 Kb daf 44132.5 13269.5 1 Kb F11 535 1687.5 133860.5 background = varies from 100 to 2500 ¹Laser power set at 80% andphotomultiplier tube set at 80%

TABLE 13 Compounds.

What is claimed is:
 1. An activated slide comprising a support surfacecoated with a bound composition of a reagent composition, the boundcomposition comprising a polymeric backbone having more than one pendentthermochemically amine-reactive or sulfhydryl-reactive groups configuredand arranged to form covalent bonds with fundamental groups on a targetmolecule, the reagent composition being coated and immobilized on thesurface in a manner that permits: a) small sample volume of a solutioncontaining the target molecule to be applied in the form of a discretespot on the reagent composition-coated surface, b) target moleculepresent in the sample volume to become attached to the bound compositionby reaction between the functional groups of the target molecule and thethermochemically amine-reactive or sulfhydryl-reactive groups of thebound composition, and c) substantially all unattached target moleculeto be washed from the sport without undue detectable amounts of targetmolecule in the area surrounding the spot, and wherein the boundcomposition is configured and arranged to form covalent bonds withfunctional groups on the target molecule without the use of attractinggroups to attract the target molecule to the bound composition.
 2. Anactivated slide according to claim 1 wherein the activated slide isadapted for fabricating a microarray wherein the target moleculecomprises a nucleic acid and the surface comprises the surface of aplastic, silicon hydride, or organosilane-pretreated glass or siliconeslide.
 3. An activated slide according to claim 2 wherein the nucleicacid comprises one or more functional groups selected from the groupconsisting of amine and sulfhydryl groups.
 4. An activated slideaccording to claim 1 wherein the activated slide is configured andarranged to receive a sample in an amount of twenty nanoliters or less.5. An activated slide according to claim 1 wherein the reagentcomposition comprises one or more latent reactive groups comprisingphotoreactive groups for attaching the reagent composition to thesurface of the slide upon application of energy from a suitable source,wherein the latent reactive groups immobilize the composition onto theslide.
 6. An activated slide according to claim 1 wherein the reagentcomposition comprises thermochemically reactive groups and photoreactivegroups, wherein the thermochemically reactive groups and thephotoreactive groups are pendent from the polymeric backbone and thephotoreactive groups are selected from the group consisting ofphotoreactive aryl ketones.
 7. An activated slide according to claim 6wherein the photoreactive aryl ketones are each, independently, selectedfrom the group consisting of acetophenone, benzophenone, anthraquinone,anthrone, and heterocyclic analogs of anthrone.
 8. An activated slideaccording to claim 1 wherein the polymeric backbone is selected from thegroup consisting of acrylics, vinyls, nylons, polyurethanes andpolyethers, the pendent thermochemically reactive groups are selectedfrom the group consisting of activated esters, epoxides, azlactones,activated hydroxyls, aldehydes, isocyanates, thioisocyanates, carboxylicacid chlorides, alkyl halides, maleimide, and α-iodoacetamide, and thebackbone further comprises one or more pendent photoreactive groupsselected from the group consisting of aryl ketones.
 9. An activatedslide for binding target molecule in a sample, the slide comprising: a.a support surface; and b. a bound composition attached to the supportsurface, wherein the bound composition comprises a polymeric backbonehaving more than one thermochemically amine-reactive orsulfhydryl-reactive groups attache thereto, and wherein the boundcomposition is configured and arranged to form covalent bonds withfunctional groups on the target molecule without use of attractinggroups to attract the target molecule to the bound composition.
 10. Anactivated slide according to claim 9 wherein the activated slide isadapted for fabricating a microarray wherein the target moleculecomprises a nucleic acid and the surface comprises the surface of aplastic, silicon hydride, or organosilane-pretreated glass or siliconeslide.
 11. An activated slide according to claim 10 wherein the nucleicacid comprises one or more functional groups selected from the groupconsisting of amine and sulfhydryl groups.
 12. An activated slideaccording to claim 9 wherein the activated slide is configured andarranged to receive a sample in an amount of twenty nanoliters or less.13. An activated slide according to claim 9 wherein the boundcomposition comprises a reaction product of a reagent composition withthe surface, wherein the reagent composition comprises one or morephotoreactive groups for attaching the reagent composition to thesurface of the slide upon application of energy from a suitable source,wherein the photoreactive groups immobilize the reagent composition ontothe slide to form the bound composition.
 14. An activated slideaccording to claim 13 wherein the reagent composition comprisesthermochemically reactive groups and photoreactive groups, wherein thethermochemically reactive and photoreactive groups are pendent from oneor more hydrophilic polymeric backbones and the photoreactive groups arephotoreactive aryl ketones.
 15. An activated slide according to claim 14wherein the photoreactive aryl ketones are each, independently, selectedfrom the group consisting of acetophenone, benzophenone, anthraquinone,anthrone, and heterocyclic analogs of anthrone.
 16. An activated slideaccording to claim 9 wherein the polymeric backbone is selected from thegroup consisting acrylics, vinyls, nylons, polyurethanes and polyethers,the pendent thermochemically reactive groups are selected from the groupconsisting of activated esters, epoxides, azlactones, activatedhydroxyls, aldehydes, isocyanates, thioisocyanates, carboxylic acidchlorides, alkyl halides, maleimide, and α-iodoacetamide, and thebackbone further comprises one or more pendent photoreactive groupsselected from the group consisting of aryl ketones.